Journal: bioRxiv

Article Title: Integration of Patient-Derived Organoids and Organ-on-Chip Systems: Investigating Colorectal Cancer Invasion within the Mechanical and GABAergic Tumor Microenvironment

doi: 10.1101/2023.09.14.557797

Figure Lengend Snippet: (A.) Schematic representing the initial events of the metastatic cascade that can be measured using the CRC-OOC. Tumor cells in the top channel (1) can be visualized and analyzed separately from tumor cells that have invaded and adhered in the endothelial compartment (2). Additionally, tumor cells that are found in the endothelial effluent (3; circulating tumor cells (“CTC-like” cells)) can also be collected and analyzed. (B.) Circulating tumor cells were collected from the endothelial effluent of stretched and not stretched HCT116 CRC-Chips and RNAseq was performed. GO Pathway analysis was performed on a subset of genes with either a 2-fold difference between stretched and not stretched CTCs or an FDR-adjusted p-value <0.1. N=2 biological replicates with 3 pooled chips in each replicate. (C.) Gene expression of neurotransmitter-related genes were measured by a neurotransmitter-specific PCR array. HCT116 tumor cells of stretched and not stretched chips were harvested on day 6 from the top epithelial channel and isolated via FACs. Data is displayed as the gene expression fold change of stretch versus not stretch conditions Expression was normalized to the average of 5 housekeeping genes. Genes that had a 1.5-fold increase or decrease in the stretched condition are displayed. N=3 biological replicates with 3 chips pooled per biological replicate. (D.) Schematic of the production of GABA from glutamate by the enzyme GAD1. In this figure, experiments related to GABA are indicated in purple and experiments related to GAD1 are indicated in light blue. (E.) Representative confocal immunofluorescent images of the epithelial (top; 1) or endothelial (bottom; 2) channel of the CRC-Chips stained for GABA (purple) on day 6. Invaded HCT116 H2B-GFP stain positive for GABA, while HCT116 H2B-GFP tumor cells that are in the top channel stain weakly for GABA. DAPI stains the nuclei of Caco2 C2BBe1 cells in the top channel and endothelial cells in the bottom channel. Scale bars represent 200 μm in the top channel image and 100 μm in the bottom channel images. Top channel images are maximum projections that span a 35 μm Z-height with a 5 μm step size. Bottom channel images are maximum projections that span a 10 μm Z-height with a 5 μm step size. (F.) RNAseq analysis was performed on CRC organoids and normalized GAD1 expression is shown. N=5 independent donors with 2-3 replicates each. Individual data points are shown and mean ± SEM is displayed. Analysis between US and UP data was performed using an unpaired t-test; ***p<0.001 ( G.) CRC organoids were isolated from stretched chips and qPCR analysis of GAD1 gene expression was performed. N=5 independent doners with 3 replicates each. Individual data points are shown and mean ± SEM is displayed. Analysis between US and UP data was performed using an unpaired t-test p<0.05. ( H.) GAD1 mRNA expression from TCGA in KRAS, NRAS, or BRAF mutant primary colon cancer tumors. N=196 patients with KRAS, NRAS, or BRAF mutant tumors; N=201 patients with KRAS, NRAS, or BRAF wildtype tumors. Individual data points are shown and median with interquartile range is represented. Data was analyzed with an unpaired t-test; ****p<0.0001. ( I.) Kaplan-Meier curve with univariate analysis of the survival of patients with KRAS, NRAS, or BRAF mutated CRC tumors based on high versus low expression of GAD1 (defined as above or below the median GAD1 mRNA expression z-score of 0.3). Data was extracted from the TCGA. N=254 patients. Data was analyzed using a log-rank (Mantel-Cox test). ( J.) Effluent from the epithelial channel of the patient-derived organoids was collected on day 0 (D0) and day 6 (D6). GABA intensity was analyzed from extracted metabolites N=6 chips per timepoint per patient; n=4 on D0 and 2 on D6 for UK. Data was analyzed using a two-way ANOVA; ***p<0.001; ****p<0.0001. ( K .) US-H2B-GFP (top) and UP-H2B-GFP (bottom) stretched tumor-chips were stained for GABA (purple). Scale bars represent 200 μm. L. Representative 10x immunofluorescence images of the 5 tumors stained for EpCAM (green), CK20 (red), and GABA (purple). Scale bars represent 500 μm and 200 μm for UK. All schematics were made in or are from BioRender.

Article Snippet: ABAT human shRNA lentiviral particles (Santa Cruz Biotechnology, Inc., #sc-93288-V) and control shRNA lentiviral particles (Santa Cruz Biotechnology, Inc., #sc-108080) were used to produce HCT116 H2B-GFP ABAT knock down cells at a multiplicity of infection (MOI) of 5 and 2 mg/mL of puromycin (Santa Cruz Biotechnology, #sc-108071) was used to select for knocked down cells.

Techniques: Gene Expression, Isolation, Expressing, Staining, Mutagenesis, Derivative Assay, Immunofluorescence

Journal: bioRxiv

Article Title: Integration of Patient-Derived Organoids and Organ-on-Chip Systems: Investigating Colorectal Cancer Invasion within the Mechanical and GABAergic Tumor Microenvironment

doi: 10.1101/2023.09.14.557797

Figure Lengend Snippet: (A.) Invasion of HCT116 tumor-chips in the presence or absence of exogenous GABA (flowed through the epithelial channel) was measured on day 6 (D6) of the experiment and normalized to day 0 (D0) invasion. N=6 chips. Individual data are shown, with mean ± SEM represented and analyzed using a one-way ANOVA; ****p<0.0001. (B.) Intracellular [ 13 C 4 ]GABA or unlabeled GABA was measured via mass spectrometry-based metabolomics in the HCT116 tumor-chips after the addition of exogenous GABA for six days. N=3 chips. (C.) Schematic of GABA catabolism by ABAT, subsequent entry into the TCA cycle, and inhibition of ABAT activity by vigabatrin. In this figure, experiments related to GABA are indicated as purple, and experiments related to ABAT are indicated as teal. (D.) Western blot analysis of ABAT in shRNA control or ABAT shRNA HCT116 tumor cells. Cropped western blot (left) and quantification (right) confirm knockdown of ABAT. (E.) Growth rate of ABAT-knockdown or control HCT116 tumor cells when grown in traditional cell culture methods. N=3. Individual data are shown and mean ± SEM are represented. Data was analyzed using a t-test; **p<0.01. (F.) Numbers of ABAT-knockdown or control HCT116 tumor cells in the top channel on-chip as measured via fluorescence microscopy and quantified on day 0 (D0) and day 6 (D6). N=5-6 chips. Individual data are shown and mean ± SEM are represented. Data was analyzed using a two-way ANOVA; *p<0.05;***p<0.001. (G.) Invasion of ABAT knockdown (KD) or control shRNA HCT116 tumor-chips in the presence or absence of stretching was measured on day 6 (D6) of the experiment and normalized to day 0 (D0) invasion. N=5-6 chips. Individual data points are shown and mean ± SEM are represented. Data was analyzed using a one-way ANOVA; *p<0.05. (H.) Numbers of HCT116 tumor cells in the top channel on-chip in the presence or absence of stretching, with or without vigabatrin was measured via fluorescence microscopy and quantified on day 0 (D0) and day 6 (Day 6). Individual data are shown and mean ± SEM are represented. N=4 chips. Data was analyzed using a two-way ANOVA; **p<0.01. (I.) Invasion of HCT116 tumor-chips in the presence or absence of stretching, with or without vigabatrin was measured on day 6 (D6) of the experiment and normalized to day 0 (D0) invasion. N=4 chips. Individual data points are shown and mean ± SEM are represented. Data was analyzed using a one-way ANOVA; ***p<0.001. (J.) Numbers of US-H2B-GFP (red) or UP-H2B-GFP (blue) tumor cells in the top channel on-chip in the presence or absence of stretching, with or without vigabatrin was measured via fluorescence microscopy and quantified on day 0 (D0) and day 6 (Day 6). N=4-5 chips. Individual data are shown and mean ± SEM are represented. Data was analyzed using a two-way ANOVA; ns=p>0.05. (K.) Invasion of US-H2B-GFP or UP-H2B-GFP organoid-tumor chips in the presence or absence of stretching, with or without vigabatrin was measured on day 6 (D6) of the experiment and normalized to day 0 (D0) invasion. N=4-5 chips. Individual data are shown and mean ± SEM are represented. Data was analyzed using a one-way ANOVA; **p<0.01. All schematics were made in or are from BioRender.

Article Snippet: ABAT human shRNA lentiviral particles (Santa Cruz Biotechnology, Inc., #sc-93288-V) and control shRNA lentiviral particles (Santa Cruz Biotechnology, Inc., #sc-108080) were used to produce HCT116 H2B-GFP ABAT knock down cells at a multiplicity of infection (MOI) of 5 and 2 mg/mL of puromycin (Santa Cruz Biotechnology, #sc-108071) was used to select for knocked down cells.

Techniques: Mass Spectrometry, Inhibition, Activity Assay, Western Blot, shRNA, Control, Knockdown, Cell Culture, Fluorescence, Microscopy

Journal: Cell reports

Article Title: PRRC2B modulates oligodendrocyte progenitor cell development and myelination by stabilizing Sox2 mRNA.

doi: 10.1016/j.celrep.2024.113930

Figure Lengend Snippet: Figure 1. PRRC2B deficiency in oligodendroglia cells leads to developmental hypomyelination (A) Immunostaining of PRRC2B/PDGFRa/OLIG2 in the corpus callosum (CC) of mice with indicated genotypes. Scale bars, 25 mm. (B) Left: western blotting analysis of the protein levels of PRRC2B in the cerebral cortex from Prrc2bf/f and Prrc2bf/f; Olig2Cre+/ mice at P6. Right: quantification of PRRC2B protein levels. Values are shown as means ± SEMs, *p < 0.05, unpaired two-tailed Student’s t test (n = 3 mice). (C) Quantitative real-time PCR detected the mRNA levels of Prrc2b in the cerebral cortex of P6 mice. Values are means ± SEMs. ***p < 0.001, unpaired two-tailed Student’s t test (each group, n = 9 replicates from >3 mice). (D) Pictures of Prrc2bf/f and Prrc2bf/f; Olig2Cre+/ mice and brains at P15. Scale bars, 5 mm (top), 2 cm (bottom).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER 2X SanTaq PCR Master Mix Sangon Biotech Cat# B532061 Trypsin Inhibitor, Soybean Sangon Biotech Cat# A003587 Papain, Suspension Sangon Biotech Cat# A003124 Hoechst33342 Sangon Biotech Cat# E607328 2x RealStar Green Fast Mixture with ROX II Genstar Cat# A304-10 Recombinant RNasin Ribonuclease Inhibitor Promega Cat# N2518 Proteinase K, recombinant PCR Grade Roche Cat# 03115828001 HA-tag Magnetic Beads Thermo Scientific Cat# 88836 Streptavidin Beads Life Technologies Cat# 11206D L-cysteine Hydrochloride, Monohydrate Amresco Cat# 0206-500G Puromycin InvivoGen Cat# ant-pr-1 Poly-D-lysine Hydrobromide Sigma Cat# P6407 Critical commercial assays NEBNext Poly(A) mRNA Magnetic Isolation Module New England Biolabs Cat# E7490 NEBNext Magnesium RNA Fragmentation Module New England Biolabs Cat# E6150S DynabeadsTM Antibody Coupling Kit Thermo Scientific Cat# 14311D Black-Gold II Myelin Staining Kit Millipore Cat# AG105 TransScript Uni All-in-One First-Strand Cdna Synthesis SuperMix for qPCR (One-Step gDNA Removal) TransGen Biotech Cat# AU341 BCA Protein Assay Kit Beyotime Biotechnology Cat# P0009 Western LightningTM Plus Chemiluminescence Reagent PerkinElmer Cat# NEL104001EA Deposited data Raw and analyzed data (MeRIP-seq) This paper GEO: GSE243310 Raw and analyzed data (RIP-seq) This paper GEO: GSE243309 Raw and analyzed data (RNA-seq) This paper GEO: GSE222394 (NSC) and GSE222395 (OPC) Experimental models: Cell lines Oli-neu Dr. Wei Mo Gift from Dr. Wei Mo O4 Dr. Wei Mo Gift from Dr. Wei Mo GalC Dr. Wei Mo Gift from Dr. Wei Mo Experimental models: Organisms/strains Mouse: Prrc2bf/f This paper Gift from Dr. Fengchao Wang Mouse: Olig2-Cre The Jackson Laboratory Gift from Dr. Bo Xiao, Stock: 011103 Mouse: Nestin-Cre The Jackson Laboratory Stock: 003771 Mouse: Prrc2b-HA This paper Center for Excellence in Molecular Cell Science of the Chinese Academy of Sciences Mouse: Prrc2af/f This paper Gift from Dr. Fengchao Wang Mouse: C57BL/6J This paper SPF(Beijing) Biotechnology Co., Ltd. Oligonucleotides RT-qPCR Primers Sangon Biotech Table S14 Recombinant DNA pCMV-N-mCherry Beyotime Biotechnology Cat# D2711 pCMV-N-mCherry-Sox2-CDS+30-UTR-wt This paper Nanjingjinsirui Science & Technology Biology Corp (Continued on next page) Cell Reports 43, 113930, March 26, 2024 17

Techniques: Immunostaining, Western Blot, Two Tailed Test, Real-time Polymerase Chain Reaction

Journal: Cell reports

Article Title: PRRC2B modulates oligodendrocyte progenitor cell development and myelination by stabilizing Sox2 mRNA.

doi: 10.1016/j.celrep.2024.113930

Figure Lengend Snippet: Figure 2. PRRC2B promotes OL progenitor cell differentiation (A) Immunofluorescence of PDGFRa/OLIG2 in the CC of Prrc2bf/f and Prrc2bf/f; Olig2Cre+/ mice at P15 or P40. Scale bars, 30 mm. (B) Quantification of PDGFRa/OLIG2-positive OPCs in the CC of mice, with the indicated geno- types at P15 or P40. Images of brain slices from >3 mice were gained and analyzed. Values are shown as means ± SEMs, **p < 0.01, NS, not statistically significant, unpaired two-tailed Student’s t test (P15: f/f, n = 19, f/f; Olig2, n = 13; P40: f/f, n = 14, f/f; Olig2, n = 6). (C) Immunostainings of CC1/OLIG2 in the CC of Prrc2bf/f, and Prrc2bf/f; Olig2Cre+/ mice at P15 or P40. Scale bars, 30 mm. (D) Quantification of CC1/OLIG2-positive OLs in the CC of mice, with the indicated genotypes at P15 or P40. Images of brain slices from >3 mice were gained and analyzed. Values are shown as means ± SEMs, **p < 0.01, ***p < 0.001, unpaired two-tailed Student’s t test (P15: f/f, n = 18, f/f; Olig2, n = 12; P40: f/f, n = 7, f/f; Olig2, n = 6). (E) Left: western blotting analysis of OPC devel- opment-related protein in the CC of mice at P15. Right: quantification of protein levels of OLIG2, MAG, MBP, and ALDH1L1. Values are shown as means ± SEMs, *p < 0.05, NS, not statistically significant, unpaired two-tailed Student’s t test (n = 3 mice). (F) Quantitative real-time PCR detection of the mRNA levels of OPC-related genes in the CC of Prrc2bf/f and Prrc2bf/f; Olig2Cre+/ mice at P15. Values were shown as means ± SEMs, *p < 0.05, **p < 0.01, ***p < 0.001, unpaired two-tailed Stu- dent’s t test (n R 4 independent biological repli- cates for each genotype).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER 2X SanTaq PCR Master Mix Sangon Biotech Cat# B532061 Trypsin Inhibitor, Soybean Sangon Biotech Cat# A003587 Papain, Suspension Sangon Biotech Cat# A003124 Hoechst33342 Sangon Biotech Cat# E607328 2x RealStar Green Fast Mixture with ROX II Genstar Cat# A304-10 Recombinant RNasin Ribonuclease Inhibitor Promega Cat# N2518 Proteinase K, recombinant PCR Grade Roche Cat# 03115828001 HA-tag Magnetic Beads Thermo Scientific Cat# 88836 Streptavidin Beads Life Technologies Cat# 11206D L-cysteine Hydrochloride, Monohydrate Amresco Cat# 0206-500G Puromycin InvivoGen Cat# ant-pr-1 Poly-D-lysine Hydrobromide Sigma Cat# P6407 Critical commercial assays NEBNext Poly(A) mRNA Magnetic Isolation Module New England Biolabs Cat# E7490 NEBNext Magnesium RNA Fragmentation Module New England Biolabs Cat# E6150S DynabeadsTM Antibody Coupling Kit Thermo Scientific Cat# 14311D Black-Gold II Myelin Staining Kit Millipore Cat# AG105 TransScript Uni All-in-One First-Strand Cdna Synthesis SuperMix for qPCR (One-Step gDNA Removal) TransGen Biotech Cat# AU341 BCA Protein Assay Kit Beyotime Biotechnology Cat# P0009 Western LightningTM Plus Chemiluminescence Reagent PerkinElmer Cat# NEL104001EA Deposited data Raw and analyzed data (MeRIP-seq) This paper GEO: GSE243310 Raw and analyzed data (RIP-seq) This paper GEO: GSE243309 Raw and analyzed data (RNA-seq) This paper GEO: GSE222394 (NSC) and GSE222395 (OPC) Experimental models: Cell lines Oli-neu Dr. Wei Mo Gift from Dr. Wei Mo O4 Dr. Wei Mo Gift from Dr. Wei Mo GalC Dr. Wei Mo Gift from Dr. Wei Mo Experimental models: Organisms/strains Mouse: Prrc2bf/f This paper Gift from Dr. Fengchao Wang Mouse: Olig2-Cre The Jackson Laboratory Gift from Dr. Bo Xiao, Stock: 011103 Mouse: Nestin-Cre The Jackson Laboratory Stock: 003771 Mouse: Prrc2b-HA This paper Center for Excellence in Molecular Cell Science of the Chinese Academy of Sciences Mouse: Prrc2af/f This paper Gift from Dr. Fengchao Wang Mouse: C57BL/6J This paper SPF(Beijing) Biotechnology Co., Ltd. Oligonucleotides RT-qPCR Primers Sangon Biotech Table S14 Recombinant DNA pCMV-N-mCherry Beyotime Biotechnology Cat# D2711 pCMV-N-mCherry-Sox2-CDS+30-UTR-wt This paper Nanjingjinsirui Science & Technology Biology Corp (Continued on next page) Cell Reports 43, 113930, March 26, 2024 17

Techniques: Cell Differentiation, Two Tailed Test, Western Blot, Real-time Polymerase Chain Reaction

Journal: Cell reports

Article Title: PRRC2B modulates oligodendrocyte progenitor cell development and myelination by stabilizing Sox2 mRNA.

doi: 10.1016/j.celrep.2024.113930

Figure Lengend Snippet: Figure 3. PRRC2B deficiency affects OL progenitor cell differentiation (A) Immunostainings of PRRC2B/PDGFRa/OLIG2 in OPCs isolated from brain tissues of Prrc2bf/f and Prrc2bf/f; Olig2Cre+/ mice at P4. Scale bars, 50 mm. (B) Western blotting analysis of the protein levels of PRRC2B in OPC-derived cells cultured in prolifera- tion medium for 4 days and quantification of PRRC2B protein levels. Values are shown as means ± SEMs, **p < 0.01, unpaired two-tailed Student’s t test (n = 4 mice). (C) Quantitative real-time PCR analysis of the mRNA levels of Prrc2b in OPCs isolated from Prrc2bf/f and Prrc2bf/f; Olig2Cre+/ mice. Values are means ± SEMs, ***p < 0.001, unpaired two-tailed Student’s t test (n = 6 independent biological replicates for each group). (D) Western blotting analysis the protein levels of OLIG2, ALDH1L1, and MBP in OPC-derived cells cultured in differentiation medium for 4 days. (E) Quantification of OLIG2, MBP, and ALDH1L1 protein levels. Values are shown as means ± SEMs, *p < 0.05, **p < 0.01, unpaired two-tailed Student’s t test (n = 3 mice). (F) Quantitative real-time PCR detection of the mRNA levels of OL-related genes in OPCs-derived cells isolated from Prrc2bf/f and Prrc2bf/f; Olig2Cre+/

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER 2X SanTaq PCR Master Mix Sangon Biotech Cat# B532061 Trypsin Inhibitor, Soybean Sangon Biotech Cat# A003587 Papain, Suspension Sangon Biotech Cat# A003124 Hoechst33342 Sangon Biotech Cat# E607328 2x RealStar Green Fast Mixture with ROX II Genstar Cat# A304-10 Recombinant RNasin Ribonuclease Inhibitor Promega Cat# N2518 Proteinase K, recombinant PCR Grade Roche Cat# 03115828001 HA-tag Magnetic Beads Thermo Scientific Cat# 88836 Streptavidin Beads Life Technologies Cat# 11206D L-cysteine Hydrochloride, Monohydrate Amresco Cat# 0206-500G Puromycin InvivoGen Cat# ant-pr-1 Poly-D-lysine Hydrobromide Sigma Cat# P6407 Critical commercial assays NEBNext Poly(A) mRNA Magnetic Isolation Module New England Biolabs Cat# E7490 NEBNext Magnesium RNA Fragmentation Module New England Biolabs Cat# E6150S DynabeadsTM Antibody Coupling Kit Thermo Scientific Cat# 14311D Black-Gold II Myelin Staining Kit Millipore Cat# AG105 TransScript Uni All-in-One First-Strand Cdna Synthesis SuperMix for qPCR (One-Step gDNA Removal) TransGen Biotech Cat# AU341 BCA Protein Assay Kit Beyotime Biotechnology Cat# P0009 Western LightningTM Plus Chemiluminescence Reagent PerkinElmer Cat# NEL104001EA Deposited data Raw and analyzed data (MeRIP-seq) This paper GEO: GSE243310 Raw and analyzed data (RIP-seq) This paper GEO: GSE243309 Raw and analyzed data (RNA-seq) This paper GEO: GSE222394 (NSC) and GSE222395 (OPC) Experimental models: Cell lines Oli-neu Dr. Wei Mo Gift from Dr. Wei Mo O4 Dr. Wei Mo Gift from Dr. Wei Mo GalC Dr. Wei Mo Gift from Dr. Wei Mo Experimental models: Organisms/strains Mouse: Prrc2bf/f This paper Gift from Dr. Fengchao Wang Mouse: Olig2-Cre The Jackson Laboratory Gift from Dr. Bo Xiao, Stock: 011103 Mouse: Nestin-Cre The Jackson Laboratory Stock: 003771 Mouse: Prrc2b-HA This paper Center for Excellence in Molecular Cell Science of the Chinese Academy of Sciences Mouse: Prrc2af/f This paper Gift from Dr. Fengchao Wang Mouse: C57BL/6J This paper SPF(Beijing) Biotechnology Co., Ltd. Oligonucleotides RT-qPCR Primers Sangon Biotech Table S14 Recombinant DNA pCMV-N-mCherry Beyotime Biotechnology Cat# D2711 pCMV-N-mCherry-Sox2-CDS+30-UTR-wt This paper Nanjingjinsirui Science & Technology Biology Corp (Continued on next page) Cell Reports 43, 113930, March 26, 2024 17

Techniques: Cell Differentiation, Isolation, Western Blot, Derivative Assay, Cell Culture, Two Tailed Test, Real-time Polymerase Chain Reaction

Journal: Cell reports

Article Title: PRRC2B modulates oligodendrocyte progenitor cell development and myelination by stabilizing Sox2 mRNA.

doi: 10.1016/j.celrep.2024.113930

Figure Lengend Snippet: Figure 4. PRRC2B is an m6A-specific bind- ing protein in the brain (A) Schematic diagram of RNA pull-down showing that the unmethylated oligo(A) and methylated oligo-m6A RNA were used to capture potential m6A binding proteins in vitro. (B) Western blotting displaying endogenous PRRC2B and YTHDF1 pulled down in the brains of P4 mice. (C) LC-MS/MS quantification of the m6A/A ratio in RNA isolated from OPCs of Prrc2bf/f and Prrc2bf/f; Olig2Cre+/ mice. Values are means ± SEMs. Un- paired two-tailed Student’s t test (n = 4 experi- ments for each group). (D) Left: HA-tag was added to the C terminus of Prrc2b in mice. Right: western blotting of the effi- ciency of HA-tag expression in whole brains of WT and Prrc2b-HA mice at P4. (E) Schematic diagram of the RIP assay for LC-MS/ MS and sequencing. (F) Quantification of the m6A/A ratio in mRNA iso- lated from the cerebral cortex of WT and Prrc2b- HA mice at P4 by RIP. Values are means ± SEMs of 3 independent experiments, *p < 0.05, unpaired two-tailed Student’s t test (n = 3 independent biological replicates). (G) Pie chart presenting the distribution of PRRC2B-binding peaks in the 30 UTR, 50 UTR, CDS, or noncoding regions. (H) Enrichment chart depicting the proportion of PRRC2B-binding peaks in the corresponding 4 regions. (I) Binding motif identified by HOMER with PRRC2B-binding peaks (p = 1e12).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER 2X SanTaq PCR Master Mix Sangon Biotech Cat# B532061 Trypsin Inhibitor, Soybean Sangon Biotech Cat# A003587 Papain, Suspension Sangon Biotech Cat# A003124 Hoechst33342 Sangon Biotech Cat# E607328 2x RealStar Green Fast Mixture with ROX II Genstar Cat# A304-10 Recombinant RNasin Ribonuclease Inhibitor Promega Cat# N2518 Proteinase K, recombinant PCR Grade Roche Cat# 03115828001 HA-tag Magnetic Beads Thermo Scientific Cat# 88836 Streptavidin Beads Life Technologies Cat# 11206D L-cysteine Hydrochloride, Monohydrate Amresco Cat# 0206-500G Puromycin InvivoGen Cat# ant-pr-1 Poly-D-lysine Hydrobromide Sigma Cat# P6407 Critical commercial assays NEBNext Poly(A) mRNA Magnetic Isolation Module New England Biolabs Cat# E7490 NEBNext Magnesium RNA Fragmentation Module New England Biolabs Cat# E6150S DynabeadsTM Antibody Coupling Kit Thermo Scientific Cat# 14311D Black-Gold II Myelin Staining Kit Millipore Cat# AG105 TransScript Uni All-in-One First-Strand Cdna Synthesis SuperMix for qPCR (One-Step gDNA Removal) TransGen Biotech Cat# AU341 BCA Protein Assay Kit Beyotime Biotechnology Cat# P0009 Western LightningTM Plus Chemiluminescence Reagent PerkinElmer Cat# NEL104001EA Deposited data Raw and analyzed data (MeRIP-seq) This paper GEO: GSE243310 Raw and analyzed data (RIP-seq) This paper GEO: GSE243309 Raw and analyzed data (RNA-seq) This paper GEO: GSE222394 (NSC) and GSE222395 (OPC) Experimental models: Cell lines Oli-neu Dr. Wei Mo Gift from Dr. Wei Mo O4 Dr. Wei Mo Gift from Dr. Wei Mo GalC Dr. Wei Mo Gift from Dr. Wei Mo Experimental models: Organisms/strains Mouse: Prrc2bf/f This paper Gift from Dr. Fengchao Wang Mouse: Olig2-Cre The Jackson Laboratory Gift from Dr. Bo Xiao, Stock: 011103 Mouse: Nestin-Cre The Jackson Laboratory Stock: 003771 Mouse: Prrc2b-HA This paper Center for Excellence in Molecular Cell Science of the Chinese Academy of Sciences Mouse: Prrc2af/f This paper Gift from Dr. Fengchao Wang Mouse: C57BL/6J This paper SPF(Beijing) Biotechnology Co., Ltd. Oligonucleotides RT-qPCR Primers Sangon Biotech Table S14 Recombinant DNA pCMV-N-mCherry Beyotime Biotechnology Cat# D2711 pCMV-N-mCherry-Sox2-CDS+30-UTR-wt This paper Nanjingjinsirui Science & Technology Biology Corp (Continued on next page) Cell Reports 43, 113930, March 26, 2024 17

Techniques: Methylation, Binding Assay, In Vitro, Western Blot, Liquid Chromatography with Mass Spectroscopy, Isolation, Two Tailed Test, Expressing, Sequencing

Journal: Cell reports

Article Title: PRRC2B modulates oligodendrocyte progenitor cell development and myelination by stabilizing Sox2 mRNA.

doi: 10.1016/j.celrep.2024.113930

Figure Lengend Snippet: Figure 5. PRRC2B regulates OL develop- ment in an m6A modification-dependent manner (A) Distribution of m6A peaks across the length of the mRNA (50 UTR, CDS, and 30 UTR). (B) Enriched motif identified by HOMER with m6A peaks in OPCs isolated from C57BL/6J mice at P4 (p = 1e122). (C) GO terms in the biological process category enriched in transcripts with downregulated expression levels in OPCs isolated from Prrc2bf/f

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER 2X SanTaq PCR Master Mix Sangon Biotech Cat# B532061 Trypsin Inhibitor, Soybean Sangon Biotech Cat# A003587 Papain, Suspension Sangon Biotech Cat# A003124 Hoechst33342 Sangon Biotech Cat# E607328 2x RealStar Green Fast Mixture with ROX II Genstar Cat# A304-10 Recombinant RNasin Ribonuclease Inhibitor Promega Cat# N2518 Proteinase K, recombinant PCR Grade Roche Cat# 03115828001 HA-tag Magnetic Beads Thermo Scientific Cat# 88836 Streptavidin Beads Life Technologies Cat# 11206D L-cysteine Hydrochloride, Monohydrate Amresco Cat# 0206-500G Puromycin InvivoGen Cat# ant-pr-1 Poly-D-lysine Hydrobromide Sigma Cat# P6407 Critical commercial assays NEBNext Poly(A) mRNA Magnetic Isolation Module New England Biolabs Cat# E7490 NEBNext Magnesium RNA Fragmentation Module New England Biolabs Cat# E6150S DynabeadsTM Antibody Coupling Kit Thermo Scientific Cat# 14311D Black-Gold II Myelin Staining Kit Millipore Cat# AG105 TransScript Uni All-in-One First-Strand Cdna Synthesis SuperMix for qPCR (One-Step gDNA Removal) TransGen Biotech Cat# AU341 BCA Protein Assay Kit Beyotime Biotechnology Cat# P0009 Western LightningTM Plus Chemiluminescence Reagent PerkinElmer Cat# NEL104001EA Deposited data Raw and analyzed data (MeRIP-seq) This paper GEO: GSE243310 Raw and analyzed data (RIP-seq) This paper GEO: GSE243309 Raw and analyzed data (RNA-seq) This paper GEO: GSE222394 (NSC) and GSE222395 (OPC) Experimental models: Cell lines Oli-neu Dr. Wei Mo Gift from Dr. Wei Mo O4 Dr. Wei Mo Gift from Dr. Wei Mo GalC Dr. Wei Mo Gift from Dr. Wei Mo Experimental models: Organisms/strains Mouse: Prrc2bf/f This paper Gift from Dr. Fengchao Wang Mouse: Olig2-Cre The Jackson Laboratory Gift from Dr. Bo Xiao, Stock: 011103 Mouse: Nestin-Cre The Jackson Laboratory Stock: 003771 Mouse: Prrc2b-HA This paper Center for Excellence in Molecular Cell Science of the Chinese Academy of Sciences Mouse: Prrc2af/f This paper Gift from Dr. Fengchao Wang Mouse: C57BL/6J This paper SPF(Beijing) Biotechnology Co., Ltd. Oligonucleotides RT-qPCR Primers Sangon Biotech Table S14 Recombinant DNA pCMV-N-mCherry Beyotime Biotechnology Cat# D2711 pCMV-N-mCherry-Sox2-CDS+30-UTR-wt This paper Nanjingjinsirui Science & Technology Biology Corp (Continued on next page) Cell Reports 43, 113930, March 26, 2024 17

Techniques: Isolation, Expressing

Journal: Cell reports

Article Title: PRRC2B modulates oligodendrocyte progenitor cell development and myelination by stabilizing Sox2 mRNA.

doi: 10.1016/j.celrep.2024.113930

Figure Lengend Snippet: Figure 6. Sox2 is the target gene of PRRC2B during OPC development (A) Primary OPCs isolated from Prrc2bf/f and Prrc2bf/f; Olig2Cre+/ mice at P4 were exposed to ActD, and RNA was extracted at the indicated time points. Quantitative real-time PCR was per- formed to assess the half-life of Sox2 mRNA. The data are shown as means ± SEMs, *p < 0.05, **p < 0.01, unpaired two-tailed Student’s t test (T1/2(f/f) = 44.99 min; T1/2(f/f; Olig2) = 20.02 min). (B) Integrative Genomics Viewer (IGV) tracks showing the read distributions of RNA-seq (top, n = 3), PRRC2B RIP-seq (center, n = 2), and MeRIP-seq (bottom, n = 3) profiling of Sox2 gene, with significant peaks highlighted in yel- low. Dark green indicates normalized RNA-seq profiles of OPCs isolated from Prrc2b cKO mice at P4, and gray shows that of OPCs of Prrc2bf/f

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER 2X SanTaq PCR Master Mix Sangon Biotech Cat# B532061 Trypsin Inhibitor, Soybean Sangon Biotech Cat# A003587 Papain, Suspension Sangon Biotech Cat# A003124 Hoechst33342 Sangon Biotech Cat# E607328 2x RealStar Green Fast Mixture with ROX II Genstar Cat# A304-10 Recombinant RNasin Ribonuclease Inhibitor Promega Cat# N2518 Proteinase K, recombinant PCR Grade Roche Cat# 03115828001 HA-tag Magnetic Beads Thermo Scientific Cat# 88836 Streptavidin Beads Life Technologies Cat# 11206D L-cysteine Hydrochloride, Monohydrate Amresco Cat# 0206-500G Puromycin InvivoGen Cat# ant-pr-1 Poly-D-lysine Hydrobromide Sigma Cat# P6407 Critical commercial assays NEBNext Poly(A) mRNA Magnetic Isolation Module New England Biolabs Cat# E7490 NEBNext Magnesium RNA Fragmentation Module New England Biolabs Cat# E6150S DynabeadsTM Antibody Coupling Kit Thermo Scientific Cat# 14311D Black-Gold II Myelin Staining Kit Millipore Cat# AG105 TransScript Uni All-in-One First-Strand Cdna Synthesis SuperMix for qPCR (One-Step gDNA Removal) TransGen Biotech Cat# AU341 BCA Protein Assay Kit Beyotime Biotechnology Cat# P0009 Western LightningTM Plus Chemiluminescence Reagent PerkinElmer Cat# NEL104001EA Deposited data Raw and analyzed data (MeRIP-seq) This paper GEO: GSE243310 Raw and analyzed data (RIP-seq) This paper GEO: GSE243309 Raw and analyzed data (RNA-seq) This paper GEO: GSE222394 (NSC) and GSE222395 (OPC) Experimental models: Cell lines Oli-neu Dr. Wei Mo Gift from Dr. Wei Mo O4 Dr. Wei Mo Gift from Dr. Wei Mo GalC Dr. Wei Mo Gift from Dr. Wei Mo Experimental models: Organisms/strains Mouse: Prrc2bf/f This paper Gift from Dr. Fengchao Wang Mouse: Olig2-Cre The Jackson Laboratory Gift from Dr. Bo Xiao, Stock: 011103 Mouse: Nestin-Cre The Jackson Laboratory Stock: 003771 Mouse: Prrc2b-HA This paper Center for Excellence in Molecular Cell Science of the Chinese Academy of Sciences Mouse: Prrc2af/f This paper Gift from Dr. Fengchao Wang Mouse: C57BL/6J This paper SPF(Beijing) Biotechnology Co., Ltd. Oligonucleotides RT-qPCR Primers Sangon Biotech Table S14 Recombinant DNA pCMV-N-mCherry Beyotime Biotechnology Cat# D2711 pCMV-N-mCherry-Sox2-CDS+30-UTR-wt This paper Nanjingjinsirui Science & Technology Biology Corp (Continued on next page) Cell Reports 43, 113930, March 26, 2024 17

Techniques: Isolation, Real-time Polymerase Chain Reaction, Two Tailed Test, RNA Sequencing

Journal: Journal of Virology

Article Title: Identification of Chicken CD74 as a Novel Cellular Attachment Receptor for Infectious Bursal Disease Virus in Bursa B Lymphocytes

doi: 10.1128/JVI.01712-19

Figure Lengend Snippet: Identification of CD74 as a putative receptor for IBDV. (A) Affinity purification with mAb against the IBDV major capsid protein VP2 in DT40 cells, incubated with VP2 (or the empty vector as a negative control), followed by mass spectrometry analysis. Many host proteins were associated with VP2, including chicken CD74. (B and C) Interaction between VP2 and the CD74 extracellular domain detected via a coimmunoprecipitation (co-IP) assay. (B) Western blot (WB) analysis using an antibody against the HA tag showing the bands corresponding to VP2 in the Flag co-IP assay. (C) Western blot analysis using an antibody against the HA tag showing the bands corresponding to the CD74 extracellular domain in the Flag co-IP assay. (D) To determine the distribution of CD74 in organs, the heart, liver, spleen, lung, kidney, thymus, and bursa of Fabricius of uninfected SPF chickens were collected to measure CD74 transcription levels. The most abundant CD74 expression was found in the bursa. (E) To confirm the involvement of chicken CD74 in the IBDV infection process, SPF chickens were challenged by vvIBDV or PBS. Bursas were collected for RT-qPCR analysis of CD74 transcription levels. CD74 mRNAs were significantly induced by vvIBDV at 12 h p.i. (*, P < 0.05; **, P < 0.01). The arithmetic means and standard deviations for at least three independent experiments performed in duplicate are shown.

Article Snippet: Total RNA was extracted from tissues or cells using the RNeasy minikit (Qiagen, Germany), and 1 μg RNA was reverse transcribed to cDNA using the ReverTra Ace qPCR RT master mix with a genomic DNA (gDNA) remover (Toyobo, Japan) in a 20-μl reaction mixture.

Techniques: Affinity Purification, Incubation, Plasmid Preparation, Negative Control, Mass Spectrometry, Co-Immunoprecipitation Assay, Western Blot, Expressing, Infection, Quantitative RT-PCR

Journal: Journal of Virology

Article Title: Identification of Chicken CD74 as a Novel Cellular Attachment Receptor for Infectious Bursal Disease Virus in Bursa B Lymphocytes

doi: 10.1128/JVI.01712-19

Figure Lengend Snippet: CD74 knockdown suppresses IBDV replication. The expression of the CD74 Ii-2 isoform was downregulated by siRNA interference or knockdown by shRNA in DT40 cells. The vvIBDV Gx strain at an MOI of 1 was added to the CD74 siRNA interference groups or the CD74 KD cell line. Infected cells were washed with PBS at 4 h p.i., and DT40 complete medium was then added. Cells and supernatants were collected at 24, 48, and 72 h p.i. for Western blotting and qPCR and at 72 h p.i. for ELD50 analysis. (A) CD74 expression levels in DT40 cells determined by Western blotting, showing that CD74 downregulation was effective. siSc., scrambled control siRNA. (B) Western blot assays showing IBDV VP2 expression declining at 24 to 48 h p.i. (C) qPCR analysis showing IBDV copy numbers dropping off at 48 h p.i. (6.34-fold decrease compared with the siRNA negative control; *, P < 0.05). (D) ELD50 assay showing the IBDV titer being downregulated at 48 h p.i. (14.0-fold decrease compared with the siRNA negative control; *, P < 0.05). (E) CD74 mRNA level in DT40 cells determined by qPCR, indicating that CD74 knockdown was effective. (F) IBDV VP2 protein expression was downregulated significantly at 24 to 72 h p.i. in the CD74 KD groups. (G) The IBDV copy number was downregulated at 24 to 72 h p.i. (103- to 104-fold decrease compared with the wild-type [WT] control; P < 0.05). (F) ELD50 assay showing the IBDV titer being downregulated at 72 h p.i. (4.42 × 104-fold decrease compared with wild-type cells; P < 0.05). The arithmetic means and standard deviations for at least three independent experiments performed in duplicate are shown.

Article Snippet: Total RNA was extracted from tissues or cells using the RNeasy minikit (Qiagen, Germany), and 1 μg RNA was reverse transcribed to cDNA using the ReverTra Ace qPCR RT master mix with a genomic DNA (gDNA) remover (Toyobo, Japan) in a 20-μl reaction mixture.

Techniques: Knockdown, Expressing, shRNA, Infection, Western Blot, Control, Negative Control

Journal: Journal of Virology

Article Title: Identification of Chicken CD74 as a Novel Cellular Attachment Receptor for Infectious Bursal Disease Virus in Bursa B Lymphocytes

doi: 10.1128/JVI.01712-19

Figure Lengend Snippet: CD74 overexpression promotes IBDV replication. CD74 Ii-2 isoform overexpression promotes IBDV infectivity. The expression of the CD74 Ii-2 isoform was upregulated by plasmid transfection in DT40 cells. Next, vvIBDV at an MOI of 1 was used to infected the cells as described in the text. Cells and supernatants were collected at 24, 48, and 72 h p.i. for Western blotting and at 48 h p.i. for qPCR analysis. (A) CD74 Ii-2 isoform overexpression remarkably promotes IBDV replication. The IBDV copy number was increased in the overexpression groups at 72 h p.i. compared with the nonoverexpression group (2.90-fold increase; *, P < 0.05). (B) IBDV VP2 protein expression is upregulated at 48 to 72 h p.i. in the overexpression group.

Article Snippet: Total RNA was extracted from tissues or cells using the RNeasy minikit (Qiagen, Germany), and 1 μg RNA was reverse transcribed to cDNA using the ReverTra Ace qPCR RT master mix with a genomic DNA (gDNA) remover (Toyobo, Japan) in a 20-μl reaction mixture.

Techniques: Over Expression, Infection, Expressing, Plasmid Preparation, Transfection, Western Blot

Journal: Journal of Virology

Article Title: Identification of Chicken CD74 as a Novel Cellular Attachment Receptor for Infectious Bursal Disease Virus in Bursa B Lymphocytes

doi: 10.1128/JVI.01712-19

Figure Lengend Snippet: CD74 isoform Ii-2 confers to vvIBDV the ability to attach to a vvIBDV-nonpermissive cell line. (A) 293T cells (nonpermissive to vvIBDV) were transfected with a full-length chicken CD74 Ii-2 plasmid with an HA tag (or the empty vector as a negative control). Twenty-four hours after transfection, cells were incubated with vvIBDV at an MOI of 5 at 37°C for 36 h to investigate whether CD74 confers susceptibility to IBDV infection. (Top) Cells were processed for confocal analysis, using IBDV VP2 mAb and an HA tag antibody as the primary antibodies. No virus (green fluorescence) was detected in the empty vector control. (Bottom) In the CD74 Ii-2 overexpression group, CD74 (red fluorescence) accumulated on the cell membrane, colocalizing with the IBDV particles (green fluorescence). No virus was observed to enter CD74-overexpressing nonpermissive cells. CD74 could confer attachment ability but not susceptibility to IBDV infection. (B) 293T cells overexpressed HA-tagged chicken CD74 Ii-2 or the empty vector for 24 h and were then incubated with 200 μg SVPs at 4°C for 1 h. After washing with PBS 5 times, cells were processed for confocal analysis as described above. SVPs (green fluorescence) were bound to the membranes of CD74-overexpressing cells (red fluorescence) and colocalized with CD74, but no binding was observed for the empty vector group. (C and D) 293T and HeLa cells (both of which are nonpermissive to vvIBDV) were transfected with a eukaryotic expression plasmid of HA-tagged chicken CD74 Ii-2 (or the empty vector as a negative control) and maintained for 24 h under normal culture conditions. The cells were then incubated with vvIBDV at an MOI of 50 at 4°C for 1 h for the binding assay and washed with PBS 5 times to remove the unbound viruses. Cells were processed for confocal analysis using the same antibodies and procedure as the ones described above. No virus (green fluorescence) was found to bind or infect 293T (C) or HeLa (D) cells transfected with the empty vector. In contrast, IBDV (green fluorescence) colocalized with overexpressed chicken CD74 (red fluorescence) on the membrane of nonpermissive 293T (C) and HeLa (D) cells. (E and F) qPCR analysis indicating that CD74 overexpression promotes IBDV binding to 293T or HeLa cells compared with the empty vector transfection group (9.55-fold increase in 293T cells [E] and 5.07-fold increase in HeLa cells [F]; both P < 0.05). The arithmetic means and standard deviations for at least three independent experiments performed in duplicate are shown.

Article Snippet: Total RNA was extracted from tissues or cells using the RNeasy minikit (Qiagen, Germany), and 1 μg RNA was reverse transcribed to cDNA using the ReverTra Ace qPCR RT master mix with a genomic DNA (gDNA) remover (Toyobo, Japan) in a 20-μl reaction mixture.

Techniques: Transfection, Plasmid Preparation, Negative Control, Incubation, Infection, Virus, Fluorescence, Control, Over Expression, Membrane, Binding Assay, Expressing

Journal: Nature Biotechnology

Article Title: A palmitoyl transferase chemical–genetic system to map ZDHHC-specific S -acylation

doi: 10.1038/s41587-023-02030-0

Figure Lengend Snippet: a , S -acylation is mediated by ZDHHC loading of long-chain acyl-CoA derived from lipid biosynthesis followed by acyl transfer to a proximal Cys of a protein substrate and regeneration of apo-ZDHHC. The reversible cycle is closed by thioester hydrolysis by APTs. b , X-ray structure of human ZDHHC20 irreversibly inhibited by lipid mimic 2-bromopalmitate (PDB ID: 6BML ). Inset, sterically demanding residues in the ZDHHC20 lipid-binding pocket contact the acyl chain distal to the DHHC catalytic site. c , Steric complementation between a ZDHHC ‘hole’ mutant and an alkyne-tagged ‘bumped’ lipid substrate probe enables selective loading and tag transfer to ZDHHC substrates, bypassing endogenous (WT) ZDHHCs. Fluorescence visualization and chemical proteomics are enabled by bioorthogonal conjugation to multifunctional capture reagents.

Article Snippet: C-terminally Myc-HA-tagged ZDHHC20 (C-HA-D20) PCR fragment was subcloned into the PmeI and AsiSI linearized C-FLAG-D20 vector using the NEBuilder HiFi Assembly Kit (NEB, E5520S).

Techniques: Derivative Assay, Binding Assay, Mutagenesis, Fluorescence, Conjugation Assay

Journal: Nature Biotechnology

Article Title: A palmitoyl transferase chemical–genetic system to map ZDHHC-specific S -acylation

doi: 10.1038/s41587-023-02030-0

Figure Lengend Snippet: a , Fatty acid probes containing an alkynyl click-handle (blue), varying chain length L = 16, 18 or 20 heavy atoms in the chain (carbons + nitrogen) and an R ‘bump’ group (red)—Ac, c Pr or Bz. b , Two-stage pairing strategy for a designed ZDHHC20 mutant optimizes probe chain length and then bump size to match the new binding cavity, with probe activation, selectivity over ZDHHC20 WT and transfer to a known ZDHHC20 substrate (IFITM3) optimized in parallel. c – f , Bump-hole loading analysis of C-terminal FLAG-tagged ZDHHC20 WT and mutants in HEK293T cells treated with 15 μM YnPal ( c , d ) or 18-Ac ( e , f ) for 4 h (D, catalytic-dead ZDHHC20(C156S); E, empty vector; n = 3 independent biological replicates average ± s.d.). g , Probe bump-size optimization by transfer assays with HA-IFITM3 and either WT ZDHHC20 (W) or ZDHHC20(Y181G) (M) co-expression in HEK293T cells ( n = 3 independent biological replicates average ± s.d.). h , Average loading and transfer activity relative to highest fluorescent/input ratio ( n = 3 independent biological replicates average ± s.d.). i , j , Enzyme kinetics for WT ZDHHC20 and ZDHHC20(Y181G) treated with Pal-CoA ( i ) or 18-Bz-CoA ( j ) using a KDH assay ( 3 ). Michaelis–Menten plots generated from average reaction rate (NADH generated μM min −1 , n = 3 independent experiments) ± s.d. versus lipid concentration (μM). d , f , h , The two-tailed unpaired t test of Prism 9.0 was used to determine P values and noted above relevant comparisons.

Article Snippet: C-terminally Myc-HA-tagged ZDHHC20 (C-HA-D20) PCR fragment was subcloned into the PmeI and AsiSI linearized C-FLAG-D20 vector using the NEBuilder HiFi Assembly Kit (NEB, E5520S).

Techniques: Mutagenesis, Binding Assay, Activation Assay, Plasmid Preparation, Expressing, Activity Assay, Generated, Concentration Assay, Two Tailed Test

Journal: Nature Biotechnology

Article Title: A palmitoyl transferase chemical–genetic system to map ZDHHC-specific S -acylation

doi: 10.1038/s41587-023-02030-0

Figure Lengend Snippet: ( a-b ) Catalytically dead ZDHHC20 is appreciably labeled by YnPal at peripheral cysteine sites. FLAG-tagged WT and ZDHHC20[C156S] constructs were transfected in HEK293T cells and treated with the indicated concentration of YnPal for 4 h. After lysis and IP with anti-FLAG resin, samples were subjected to CuAAC with TAMRA azide and separated by SDS-PAGE. ZDHHC20 loading and input were measured by in-gel fluorescence and anti-ZDHHC20 immunoblot (n = 3 independent biological replicates). ( c-d ) Thioester dependence of ZDHHC20 labeling was demonstrated upon treatment of YnPal and C18-Bz treated samples with 0.8 M neutralized NH 2 OH following IP and CuAAC with TAMRA azide (n = 3 independent biological replicates). ( e-f ) Time-course measuring 15 µM YnPal labeling of ZDHHC20 WT expressing HEK293T cells (n = 3 independent biological replicates). ( g-h ) Labeling activity of the indicated concentrations of YnPal in FLAG-tagged ZDHHC20[Y181G] and ZDHHC20[Y181G/C156S] expressing HEK293T cells (n = 3 independent biological replicates). The average (n = 3 independent biological replicates) loading ( b , d , f and h ) was reported as a percent of the maximal fluorescent: input ratios ± S.D. between treatments with and without hydroxylamine. ( i-l ) Probe chain-length was optimized against ZDHHC20[Y181G] using cell-based loading ( i-j ) and transfer ( k-l ) assays in HEK293T using ZDHHC20 WT (W) and ZDHHC20[Y181G] (M). ( i ) HEK293T cells were treated with 15 µM acetyl bumped probes of L = 16, 18 and 20 for 4 h and enzyme loading assessed by in-gel fluorescence following anti-FLAG IP and CuAAC with TAMRA azide (n = 3 independent biological replicates). ( k ) HEK293T cells co-expressing ZDHHC20[Y181G] and HA-Ifitm3 were treated with 15 µM 18-Ac or 20-Ac for 4 h with loading and transfer of the probe assessed following by anti-FLAG/anti-HA IP and CuAAC with TAMRA azide (n = 3 independent biological replicates). ( j-l ) The average (n = 3 independent biological replicates) loading and transfer activity were reported as a percent of the maximal fluorescent/input ratios ± S.D. The two tailed unpaired t-test of Prism 9.0 was used to determine p-values and are note above relevant comparisons.

Article Snippet: C-terminally Myc-HA-tagged ZDHHC20 (C-HA-D20) PCR fragment was subcloned into the PmeI and AsiSI linearized C-FLAG-D20 vector using the NEBuilder HiFi Assembly Kit (NEB, E5520S).

Techniques: Labeling, Construct, Transfection, Concentration Assay, Lysis, SDS Page, Fluorescence, Western Blot, Expressing, Activity Assay, Two Tailed Test

Journal: Nature Biotechnology

Article Title: A palmitoyl transferase chemical–genetic system to map ZDHHC-specific S -acylation

doi: 10.1038/s41587-023-02030-0

Figure Lengend Snippet: ( a ) Wild-type (WT), Y181G (YG), C156S (CS) and Y181G/C156S (YGCS) FLAG-tagged ZDHHC20 constructs were transfected into HEK293T cells and purified by anti-FLAG agarose affinity chromatography. After enzyme elution with 3X FLAG-peptide, buffer was exchanged using 50 kDa M.W. cut-off protein concentrator tubes and sample concentration determined using a BSA standard curve. All samples were run on SDS-PAGE gels and protein visualized by Coomassie staining (n = 2 independent experiments). ( b ) An enzyme-coupled assay monitoring ZDHHC20 autoacylation was established using commercial α-ketoglutarate dehydrogenase enzyme (KDH) along with its substrates α-ketoglutarate (α-KG), thiamine pyrophosphate (TPP) and NAD+. Optimization of α-ketoglutarate dehydrogenase (KDH) ( c ) and WT ZDHHC20 ( d ) concentrations. Pal-CoA ( e ) and 18-Bz-CoA ( f ) KDH activities were determined in the absence of ZDHHC20, to establish background rates for each probe. ( g ) 18-Bz-CoA displayed significant background activity in the KDH assay without ZDHHC20. Reaction rates for ZDHHC20[C156S] ( h ) and ZDHHC20[Y181G, C156S] ( i ) treated with Pal-CoA or 18-Bz-CoA. Michaelis-Menten plots generated by plotting average (n = 3 independent experiments) reaction rates (NADH generated (µM)/min) ± S.D.) versus lipid concentration (µM) using Prism 9.0. For reactions with 18-Bz-CoA, the basal rates at all concentrations tested were subtracted from the corresponding total reaction rates.

Article Snippet: C-terminally Myc-HA-tagged ZDHHC20 (C-HA-D20) PCR fragment was subcloned into the PmeI and AsiSI linearized C-FLAG-D20 vector using the NEBuilder HiFi Assembly Kit (NEB, E5520S).

Techniques: Construct, Transfection, Purification, Affinity Chromatography, Concentration Assay, SDS Page, Staining, Activity Assay, Generated

Journal: Nature Biotechnology

Article Title: A palmitoyl transferase chemical–genetic system to map ZDHHC-specific S -acylation

doi: 10.1038/s41587-023-02030-0

Figure Lengend Snippet: FLAG-tagged ZDHHC20 WT and ZDHHC20[Y181G] expressing HEK293T cells were treated with the indicated concentration of 18-Bz ( a-b ) for 4 h in cell-based loading assays (n = 3 independent biological replicates). ( c-d ) FLAG-tagged ZDHHC20[Y181G] and HA-Ifitm3 expressing HEK293T cells were treated with 15 µM 18-Bz for the indicated time in cell-based loading and transfer assays (n = 3 independent biological replicates). ( e-f ) FLAG-tagged ZDHHC20 WT and ZDHHC20[Y181G] expressing HEK293T cells were treated with 15 µM 18-Bz for the indicated times (n = 3 independent biological replicates). Lysates were clicked with TAMRA azide then analyzed by in-gel fluorescence and SDS-PAGE; note YG-dependent labeling of substrate protein bands (*). Input was assessed by anti-ZDHHC20 (D20) immunoblot. The average (n = 3 independent biological replicates) loading ( b, d & f ) and transfer ( d ) were reported as a percent of the maximal fluorescent: input ratios ± S.D. ( g-h ) The effect of FBS concentration on ZDHHC20 loading and transfer. ( g ) FLAG-tagged wild-type (WT) or ZDHHC20[Y181G] (M) and HA-Iftim3 expressing HEK293T cells were treated with 15 µM YnPal or 18-Bz in the presence of 0.5 or 10% FBS for 4 h in cell-based transfer assays. ( h ) The average (n = 3 independent biological replicates) loading and transfer were reported as a percent of the maximal fluorescent: input ratio ± S.D. The two tailed unpaired t-test of Prism 9.0 was used to determine p-values and noted above the relevant comparisons.

Article Snippet: C-terminally Myc-HA-tagged ZDHHC20 (C-HA-D20) PCR fragment was subcloned into the PmeI and AsiSI linearized C-FLAG-D20 vector using the NEBuilder HiFi Assembly Kit (NEB, E5520S).

Techniques: Expressing, Concentration Assay, Fluorescence, SDS Page, Labeling, Western Blot, Two Tailed Test

Journal: Nature Biotechnology

Article Title: A palmitoyl transferase chemical–genetic system to map ZDHHC-specific S -acylation

doi: 10.1038/s41587-023-02030-0

Figure Lengend Snippet: ( a ) Left: representative confocal microscopy images showing average signal of Z-stacks of HEK293T cells transiently co-expressing ZDHHC20 WT HA-tagged and Y181G mutant FLAG-tagged. Each image shows signal for HA (magenta), FLAG (green), p-cadherin as plasma membrane marker (yellow), nucleus (blue) and a composite image of all signals. Scale bar at the bottom right marks 20 mm, while the other white line highlights the region of interest (ROI) used for image analysis. Right: plot showing normalized fluorescence signal for each of the channels in the ROI (1 biological replicate). ( b ) As a , but with using Gm130 as Golgi marker (1 biological replicate).

Article Snippet: C-terminally Myc-HA-tagged ZDHHC20 (C-HA-D20) PCR fragment was subcloned into the PmeI and AsiSI linearized C-FLAG-D20 vector using the NEBuilder HiFi Assembly Kit (NEB, E5520S).

Techniques: Confocal Microscopy, Expressing, Mutagenesis, Membrane, Marker, Fluorescence

Journal: Nature Biotechnology

Article Title: A palmitoyl transferase chemical–genetic system to map ZDHHC-specific S -acylation

doi: 10.1038/s41587-023-02030-0

Figure Lengend Snippet: a , Chemical proteomic OBH workflow for enrichment and identification of S -acyltransferase substrates and S -acylation sites by LC–MS/MS. b , Chemical proteomic analysis of ZDHHC20 substrates in HEK293T cells (15 µM 18-Bz, 8 h). Enrichment in ZDHHC20(Y181G) cells over WT ZDHHC20 reveals selective ZDHHC20 loading (red triangle), and significantly enriched substrates (green circles) selected for further validation (red circles), with site identification data (blue triangles; Student’s two-tailed unpaired t test, S 0 = 0.5, adjusted FDR = 0.01, n = 4 independent biological replicates per condition). c , d , LC–MS/MS spectrum corroborating reported sites of CD151 ( c ) S -acylation at Cys11 and Cys15 and of STX7 ( d ) S -acylation at Cys28 (see also Extended Data Fig. ). e , S -acylated proteome profiling using YnPal. HEK293T cells transiently transfected with WT ZDHHC20 or ZDHHC20(Y181G) were treated with 15 µM YnPal for 8 h before processing using the on-bead digestion workflow. Substrates highlighted in green had been identified using a chemical–genetic system (Student’s two-tailed unpaired t test, S 0 = 0.5, adjusted FDR = 0.01, n = 4 independent biological replicates per condition). f , g , Validation of S -acylation for substrates at endogenous levels. HEK293T cells transiently transfected with WT ZDHHC20 (W) or ZDHHC20(Y181G) (M) were treated with 15 µM 18-Bz ( f ) or 15 µM YnPal ( g ) for 24 h. Lysates were clicked with biotin azide before enrichment on neutravidin magnetic beads. Representative immunoblots are shown for input and pull-down signals ( n = 2 independent biological replicates). h , Venn diagram of putative ZDHHC20 substrates identified in HEK293T, MDA-MB231 and PANC1 cells. i , Statistical overrepresentation analysis of putative ZDHHC20 substrate cellular compartment (Slim)-GO terms compared to the full human genome using the PANTHER classification system showing terms with >9 −log ( P value) from an FDR-adjusted two-tailed Fisher’s exact test.

Article Snippet: C-terminally Myc-HA-tagged ZDHHC20 (C-HA-D20) PCR fragment was subcloned into the PmeI and AsiSI linearized C-FLAG-D20 vector using the NEBuilder HiFi Assembly Kit (NEB, E5520S).

Techniques: Liquid Chromatography with Mass Spectroscopy, Two Tailed Test, Transfection, Magnetic Beads, Western Blot

Journal: Nature Biotechnology

Article Title: A palmitoyl transferase chemical–genetic system to map ZDHHC-specific S -acylation

doi: 10.1038/s41587-023-02030-0

Figure Lengend Snippet: ( a ) Full gel and western blots of all replicates corresponding to the chemical proteomics ZDHHC20 substrate identification (Fig. ) performed in HEK293T cells. A portion of the lysate was clicked with TAMRA azide for analysis by in-gel fluorescence. The bands present at ~35 kDa in ZDHHC20[Y181G] (M) lanes but absent in WT ZDHHC20 lanes indicate selective loading of 18-Bz on ZDHHC20[Y181G] over WT-ZDHHC20. Anti-FLAG WB indicates similar expression levels of WT construct compared to ZDHHC20[Y181G] construct. Vinculin is used as loading control (n = 4 independent biological replicates). ( b-e ) Chemical proteomics ZDHHC20 substrate detection with 18-Bz probe (15 µM) in ( b ) PANC1 cells and (C) MDA-MB-231 cells. Cells were transiently transfected with WT ZDHHC20 versus ZDHHC20[Y181G] (M) then clicked with biotin azide and enriched on neutravidin agarose for proteomic processing. Significantly enriched putative substrates (Student’s two tailed unpaired t-test S0 - 0.5, adjusted FDR - 0.01) are shown as green circles, hits with site identification data are shown in as blue triangles and other validated substrates are highlighted as red circles. 200 putative ZDHHC20 substrates are identified in (B) PANC1 cells and 50 putative substrates in ( c ) MDA-MB-231 cells. ( d-e ) Gel and western blots corresponding to the volcano plot in ( a-b ) where a portion of the lysate was clicked with TAMRA azide as described in B (n = 4 independent biological replicates). (f) Statistical over/underrepresentation analysis of putative ZDHHC20 substrate biological process GO-terms compared to a reference list containing reported S -acylated proteins (SwissPalm) using the PANTHER classification system showing terms with >1.5 -Log 10 (p-value) from an FDR adjusted Fisher’s exact two tailed test. (g-h) PTRH2 Site ID analysis and quantification. (g ) Validation of HA-PTRH2 S -acylation by ZDHHC20 using the bumped probe 18-Bz and S -acylation site mutants. Representative images (n = 3 independent biological replicates) for TAMRA signal are shown, as well as for HA and FLAG immunoblots for HA pull down and input. Calnexin was used as loading control. (h ) Bar plot showing the ratio of TAMRA fluorescence and HA pulldown signal of PTRH2 cysteine mutants as a percentage of WT PTRH2 ratio. The two tailed unpaired t-test statistical module of Prism 9.0 was used to calculate p-values and noted above relevant comparisons. (i) Profiling of Flp-in T-Rex substrates ZDHHC20 cell lines. The average (n = 3 independent biological replicates) Fold change of FLAG signal is reported as a percent of the maximal ratios ± S.D. The unpaired t-test statistical module of Prism 9.0 was used to determine p-values and noted above relevant comparisons. Related to main Fig. .

Article Snippet: C-terminally Myc-HA-tagged ZDHHC20 (C-HA-D20) PCR fragment was subcloned into the PmeI and AsiSI linearized C-FLAG-D20 vector using the NEBuilder HiFi Assembly Kit (NEB, E5520S).

Techniques: Western Blot, Fluorescence, Expressing, Construct, Control, Transfection, Two Tailed Test

Journal: Nature Biotechnology

Article Title: A palmitoyl transferase chemical–genetic system to map ZDHHC-specific S -acylation

doi: 10.1038/s41587-023-02030-0

Figure Lengend Snippet: ( a-b ) Gels and corresponding volcano plots for HEK293T cells treated with 15 μM 20- c Pr for 8 h in the presence of ZDHHC15 WT or ZDHHC15[Y184G]. ( a ) Lysates were subjected to CuAAC with TAMRA azide to assess probe incorporation and expression levels of FLAG-tagged ZDHHC and the loading control vinculin. ( b ) Volcano plot showing enrichment of putative ZDHHC15 substrates by ZDHHC15[Y184G] (Student’s two tailed unpaired T-test, S 0 0.5, adjusted FDR 0.01, n = 4 independent biological replicates) of matched lysates processed by OBH workflow and analyzed by LC-MS/MS. The positive control ZDHHC15 (red triangle) shows enrichment and many sites of modification (blue triangle) were identified through our OBH workflow. ( c-d ) Gels and corresponding volcano plots for PANC1 cells treated as described in ( a-b ). ( e-f ) Gels and corresponding volcano plots for HEK293T cells treated with 15 μM 20- Bz for 8 h in the presence of ZDHHC7 WT or ZDHHC7[L57G]. ( e ) Lysates were subjected to CuAAC with TAMRA azide to assess probe incorporation and expression levels of FLAG-tagged ZDHHCs and the loading control vinculin. ( f ) Volcano plot showing enrichment of putative ZDHHC7 substrates by ZDHHC7[L57G] (Student’s two tailed unpaired T-test, S 0 0.5, adjusted FDR 0.05, n = 4 independent biological replicates) of matched lysates processed by OBH workflow and analyzed by LC-MS/MS. ( g-h ) Overlap among ZDHHC substrate profiles for ZDHHC7, ZDHHC15, and ZDHHC20 (Student’s two tailed unpaired t-test, S 0 0.5, adjusted FDR 0.05, n = 4 independent biological replicates). ( g ) Volcano plot of ZDHHC20 OBH shown in Fig. , with unique putative substrates; putative substrates shared with ZDHHC7 and/or ZDHHC15 highlighted. ( h ) Volcano plot of ZDHHC7 OBH shown in Supplementary Fig. with unique putative substrates; putative substrates shared with ZDHHC15 and/or ZDHHC20 highlighted.

Article Snippet: C-terminally Myc-HA-tagged ZDHHC20 (C-HA-D20) PCR fragment was subcloned into the PmeI and AsiSI linearized C-FLAG-D20 vector using the NEBuilder HiFi Assembly Kit (NEB, E5520S).

Techniques: Expressing, Control, Two Tailed Test, Liquid Chromatography with Mass Spectroscopy, Positive Control, Modification

Journal: Nature Biotechnology

Article Title: A palmitoyl transferase chemical–genetic system to map ZDHHC-specific S -acylation

doi: 10.1038/s41587-023-02030-0

Figure Lengend Snippet: a , b , ZDHHC20(Y181G) retains exquisite selectivity for specific cysteines on substrates IFITM3 ( a ) and PI4K2A ( b ; n = 3 independent biological replicates average ± s.d.), matching previously reported labeling, with the 18-Bz bumped probe. c , Validation of HA-STX7 S -acylation by ZDHHC20 using the bumped probe 18-Bz and S -acylation site mutants (C28A) and (C239A). Representative images ( n = 3 independent biological replicates average ± s.d.) for TAMRA signal are shown, as well as for HA and FLAG immunoblots for HA pull down and input. Calnexin was used as loading control. d , e , Validation of HA-VAMP3 and HA-BCAP31 site S -acylation by ZDHHC20 using the bumped probe 18-Bz and S -acylation site mutants, VAMP3(C76A) and BCAP31(C23A). d , Cell-based transfer assays were performed without FLAG-ZDHHC20 and HA-VAMP3 enrichment, but rather with direct labeling of cell lysates by TAMRA-azide click followed by SDS–PAGE and anti-HA, anti-FLAG and anti-vinculin immunoblot analysis. e , FLAG-ZDHHC20 and HA-BCAP31 constructs were enriched before TAMRA-azide click labeling. f , Confirmation of trans -auto- S -acylation in peripheral cysteines on a catalytically dead C-HA-ZDHHC20(C156S) (D) by a mutant C-FLAG-ZDHHC20(Y181G) (M) with 15 μM 18-Bz. Catalytically dead C-FLAG-ZDHHC20(Y181G) (DM) did not transfer the probe to D. Cells transfected with an empty vector (E) were used as negative control. HA- and FLAG-tagged ZDHHC20 constructs were transiently cotransfected into HEK293T cells and treated with 15 μM 18-Bz for 4 h. After cell lysis, constructs were separately enriched on anti-HA and anti-FLAG resins, clicked with TAMRA-azide and separated by SDS–PAGE. Loading and input were visualized by in-gel fluorescence and immunoblot, respectively. The average ( n = 3 independent biological replicates) loading and transfer activity were reported as a percent of the maximal fluorescent:input ratios ± s.d. a , c , f , The two-tailed unpaired t test of Prism 9.0 was used to determine P values and noted above relevant comparisons.

Article Snippet: C-terminally Myc-HA-tagged ZDHHC20 (C-HA-D20) PCR fragment was subcloned into the PmeI and AsiSI linearized C-FLAG-D20 vector using the NEBuilder HiFi Assembly Kit (NEB, E5520S).

Techniques: Labeling, Western Blot, Control, SDS Page, Construct, Mutagenesis, Transfection, Plasmid Preparation, Negative Control, Lysis, Fluorescence, Activity Assay, Two Tailed Test

Journal: Nature Biotechnology

Article Title: A palmitoyl transferase chemical–genetic system to map ZDHHC-specific S -acylation

doi: 10.1038/s41587-023-02030-0

Figure Lengend Snippet: a , Profile of WT ZDHHC20 (W) or ZDHHC20(Y181G) Flp-In 293 T-REx cell lines treated with 18-Bz (15 µM, 24 h). Lysates were clicked with TAMRA azide and then analyzed by in-gel fluorescence and SDS–PAGE. Note that the asterisk represents YG-dependent labeling of substrate protein bands. b , Comparison of protein expression levels between doxycycline induction of Flp-In 293 T-REx cells and overexpression by transient expression in HEK293T cells. Representative immunoblots are shown for FLAG at high or low exposure, to probe for ZDHHC20 WT versus ZDHHC20(Y181G), and calnexin as loading control ( n = 3 independent biological replicates). c , In Flp-In 293 T-REx cells ZDHHC20(Y181G) retains exquisite selectivity for its substrate IFITM3 with the 18-Bz bumped probe, as seen in prior experiments. d , Chemical proteomic analysis of ZDHHC20 substrates in Flp-In 293 T-REx cells (15 µM 18-Bz, 24 h). Enrichment in T-REx ZDHHC20(Y181G) cells over T-REx WT ZDHHC20 reveals selective ZDHHC20 modification of substrates (green) (Student’s two-tailed unpaired t test, S 0 = 0.5, adjusted FDR = 0.01, n = 4 independent biological replicates per condition). e , f , Validation of S -acylation for T-REx ZDHHC20(Y181G) substrates at endogenous levels. Flp-In 293 T-REx cells, WT ZDHHC20 (W) or ZDHHC20(Y181G) (M), induced with doxycycline for 24 h, were treated with 15 µM 18-Bz ( e ) or YnPal ( f ) for 24 h. Lysates were clicked with biotin azide before enrichment on neutravidin magnetic beads. Representative immunoblots are shown for input and pull-down signals ( n = 2 independent replicates).

Article Snippet: C-terminally Myc-HA-tagged ZDHHC20 (C-HA-D20) PCR fragment was subcloned into the PmeI and AsiSI linearized C-FLAG-D20 vector using the NEBuilder HiFi Assembly Kit (NEB, E5520S).

Techniques: Fluorescence, SDS Page, Labeling, Comparison, Expressing, Over Expression, Western Blot, Control, Modification, Two Tailed Test, Magnetic Beads

Journal: Nature Biotechnology

Article Title: A palmitoyl transferase chemical–genetic system to map ZDHHC-specific S -acylation

doi: 10.1038/s41587-023-02030-0

Figure Lengend Snippet: ( a ) Untreated (UT) or gRNA/CAS9 treated (pSpCas9(BB)-2A-Puro, PX459 plasmid) HEK293T cells were probed with anti-ZDHHC20 (D20) and –vinculin antibodies. Cells treated with gRNA1/CAS9 resulted in knockdown (KD); whereas cells treated with gRNA2/CAS9 yielded two ZDHHC20-knockout (D20-KO) clones: KO1 and KO2 (n = 2 independent biological replicates). ( b ) WT or KO2 HEK293T cells were transfected with HA-IFITM3 and empty vector or C-FLAG-tagged ZDHHC20. Cells were then treated with 15 mM YnPal for 4 h before being harvested and lysed. IFITM3 and D20 were enriched in one pot with a mix of anti-HA and –FLAG resins before being treated with TAMRA-azide and click reagents. Tagged proteins were eluted from beads with 1X Laemmli buffer and separated by SDS-PAGE. YnPal ZDHHC20-loading and transfer to IFITM3 and input were visualized by in-gel fluorescence and anti-HA and -FLAG immunoblot, respectively (n = 2 independent biological replicates). ( c ) The average (n = 3 independent biological replicates) loading and transfer activity was reported as a percent of the maximal D20 fluorescent: input ratio and as a percent of the WT IFITM3 (empty vector) fluorescent: input ratio ± S.D. The two tailed unpaired t-test of Prism 9.0 was used to determine p-values and noted above relevant comparisons ( d-g ) WT HEK293T cells, two ZDHHC20 KO clones, and one partial knockdown (KD) clone were treated with 15 μM YnPal for 8 h. As a control for lipidation, HEK293T cells were treated with palmitic acid (Pal) and also taken through the experiment. Samples were then clicked with biotin-TAMRA-azide, 10% of which was analyzed by SDS-PAGE, in-gel fluorescence, and anti-tubulin western blot ( d ) (n = 3 independent biological replicates). The remainder was enriched on dimethylated neutravidin beads and digested for LC-MS/MS LFQ analysis. (E-G) Whilst a small number of proteins are identified as being significantly enriched/depleted (Student’s two tailed unpaired T-test S 0 – 0.1, adjusted FDR – 0.05), they are few in number and none are consistently found which correspond to our putative chemical genetic substrates found in HEK293T cells. ( f ) Analysis of YnPal treated cells against Pal shows a large number a potentially lipidated proteins have been identified, with many well validated S -acylation proteins identified, some of which have been highlighted in blue.

Article Snippet: C-terminally Myc-HA-tagged ZDHHC20 (C-HA-D20) PCR fragment was subcloned into the PmeI and AsiSI linearized C-FLAG-D20 vector using the NEBuilder HiFi Assembly Kit (NEB, E5520S).

Techniques: Plasmid Preparation, Knockdown, Knock-Out, Clone Assay, Transfection, SDS Page, Fluorescence, Western Blot, Activity Assay, Two Tailed Test, Control, Liquid Chromatography with Mass Spectroscopy

Journal: Nature Biotechnology

Article Title: A palmitoyl transferase chemical–genetic system to map ZDHHC-specific S -acylation

doi: 10.1038/s41587-023-02030-0

Figure Lengend Snippet: ( a ) Schematic representation of TurboID fusion proteins used for proximity labeling experiments. ( b-c ) Confirmation of the expression of each fusion protein by western blot after generation of ‘Jump-in’ cell lines using either anti-V5 antibody ( b ) (n = 2 independent biological replicates) or an anti-GFP antibody ( c ) (n = 2 independent biological replicates). The labeling efficiency of the TurboID biotin ligase was confirmed by the addition of 500 μM biotin for the indicated times. Only those cells expressing the ligase show an increase in the biotinylation of proteins, as determined by Streptavidin conjugated HRP, compared to the UT HEK293T cells, and also in a time dependent manner. ( d ) Volcano plot showing the enrichment of proteins when comparing the C-terminally tagged ZDHHC20 with the N-terminally tagged construct (Student’s two tailed unpaired t-test S 0 – 0.1, adjusted FDR 0.01). There does appear to be a preference for either the N- or C- terminus for some interactors, none of these correspond to our chemical genetic hits. ( e ) Volcano plot showing the enrichment of proteins when comparing the N-terminally tagged ZDHHC20 with the Turbo GFP construct (Student’s two tailed unpaired t-test S 0 – 0.1, adjusted FDR 0.01). ( f ) TurboID-based proximity-labeing enabled detection of ZDHHC20 (D20) interactors. Volcano plot showing the mean log 2 difference in protein group intensities between N-TurboID-ZDHHC20 and TurboID-GFP clones (Student’s two tailed unpaired T-test S 0 – 0.1, adjusted FDR 0.01).

Article Snippet: C-terminally Myc-HA-tagged ZDHHC20 (C-HA-D20) PCR fragment was subcloned into the PmeI and AsiSI linearized C-FLAG-D20 vector using the NEBuilder HiFi Assembly Kit (NEB, E5520S).

Techniques: Labeling, Expressing, Western Blot, Construct, Two Tailed Test, Clone Assay

Journal: Nature Biotechnology

Article Title: A palmitoyl transferase chemical–genetic system to map ZDHHC-specific S -acylation

doi: 10.1038/s41587-023-02030-0

Figure Lengend Snippet: a , Structure-guided ZDHHC engineering exemplified for ZDHHC7 (see also Extended Data Figs. and ). ZDHHC7 homology model (yellow/orange) overlayed on experimental ZDHHC20 structure (dark green) identifies a potential hole-generating amino acid (Leu57) on an adjacent helix in the vicinity of ZDHHC20 Tyr181; lipid density (blue mesh), and length/size probe analysis identifies a mutant/probe pair (ZDHHC7(L57G)/20-Bz) with optimal activity and selectivity over WT ZDHHC7. b , Bump-hole analysis of N-FLAG-tagged WT ZDHHCs or mutant ZDHHCs ZDHHC3(I182G) (D3), ZDHHC7(L57G) (D7), ZDHHC11(M181A) (D11) and ZDHHC15(Y184G) (D15) in HEK293T cell-based loading assays using 15 µM corresponding optimized probe. c , Average ( n = 3 independent biological replicates) loading reported as a percent of maximal fluorescent:input ratio ± s.d. P values determined by Prism 9.0 two-tailed unpaired t test statistical module and noted above relevant comparisons. d , ZDHHC15 substrate discovery in HEK293T cells treated with 15 µM 20- c Pr in HEK293T cells using the OBH workflow. In total, 107 chemical–genetic ZDHHC15 substrates were identified (Student’s two-tailed unpaired t test, S 0 = 0.5, adjusted FDR = 0.01, n = 4 independent biological replicates). Substrates unique or in common with parallel analyses for DHHC7 and DHHC20 in HEK293T cells are highlighted (Extended Data Fig. ). e , Overlap of chemical–genetic ZDHHC substrates identified in HEK293T cells. Of 301 total substrates, only 87 are shared by 2 or more family members, suggesting distinct substrate pools for each ZDHHC.

Article Snippet: C-terminally Myc-HA-tagged ZDHHC20 (C-HA-D20) PCR fragment was subcloned into the PmeI and AsiSI linearized C-FLAG-D20 vector using the NEBuilder HiFi Assembly Kit (NEB, E5520S).

Techniques: Mutagenesis, Activity Assay, Two Tailed Test

Journal: Cell reports

Article Title: Pten regulates endocytic trafficking of cell adhesion and Wnt signaling molecules to pattern the retina

doi: 10.1016/j.celrep.2024.114005

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Micro BCA TM Protein Assay Kit , ThermoFisher Scientific , Cat# #23235 RRID: N/A.

Techniques: Control, Affinity Purification, Recombinant, Bicinchoninic Acid Protein Assay, Western Blot, Electron Microscopy, RNAscope, Multiplex Assay, Mass Spectrometry, Mutagenesis, Software, Microscopy

Journal: eLife

Article Title: A bidirectional switch in the Shank3 phosphorylation state biases synapses toward up- or downscaling

doi: 10.7554/elife.74277

Figure Lengend Snippet: Figure 2. Neuronal activity bidirectionally modulates the phosphorylation state of Shank3. (A) The experiment protocol for extraction of Shank3 from rat cultured neocortical neurons for further quantitative mass spectrometry (MS) or Western blot analyses. (B) Volcano plot of quantitative MS data showing Shank3 residues that were differentially phosphorylated in tetrodotoxin (TTX)-treated samples compared to untreated controls. The log2 values of fold changes, if below zero, indicated hypophosphorylation (paired t-test: S1586, adjusted p=0.034142, S1614/5, 0.014444). (C) Top: diagram showing the location of S1586 and S1615 within the rat Shank3 protein. Functional domains: ANK = ankyrin repeat; SH3 = SRC homology 3; PDZ = PSD-95/Disc Large/ZO-1; Pro-rich = proline rich; SAM = sterile alpha motif. Bottom: homology comparison of sequences flanking rat S1586 and S1615 (matching mouse S1539) across species (human Shank3: NP_001358973.1; rat Shank3: NP_067708.2; mouse Shank3: UniprotKB: Q4ACU6.3). Phosphosites of interest are labeled in red; the only residue not conserved is shown in blue. (D, E) Representative Western blot using an antibody specific for phosphorylated S1615, showing changes in Shank3 phosphorylation after 10 min (D) or 24 hr (E) treatment with TTX or picrotoxin (PTX). (F) Quantification of the fold change of Shank3 S1615 phosphorylation in (D). Dashed line indicates the baseline untreated control (one-sample t-test: TTX, ***p=0.0005, PTX, **p=0.0035, n = 5 and 10 biological replicates, respectively). (G) Quantification of the fold change of Shank3 S1615 phosphorylation in (E) (one-sample t-test: TTX, ****p<0.0001, PTX, p = 0.6336, n = 7 and 7 biological replicates, respectively). Solid colored horizontal

Article Snippet: DOI: https://doi.org/10.7554/eLife.74277 19 of 31 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Recombinant DNA reagent pAAV- CMV- PI- EGFP- WPRE- bGH Gift from James M. Wilson Addgene# 105530; RRID:Addgene_105530 Commercial assay or kit Lipofectamine 2000 Thermo Fisher Scientific Cat# 11668- 027 Commercial assay or kit Gibson Assembly Master Mix New England Biolabs Cat# E2611S Commercial assay or kit Lambda protein phosphatase New England Biolabs Cat# P0753S Commercial assay or kit BCA Protein Assay Kit Thermo Fisher Scientific Cat# 23227 Commercial assay or kit Protein- G Magnetic Beads Thermo Fisher Scientific Cat# 88847 Commercial assay or kit SimplyBlue SafeStain Thermo Fisher Scientific Cat# LC6060 Commercial assay or kit PP2A Immunoprecipitation Phosphatase Assay Kit Millipore Cat# 17- 313 Commercial assay or kit Ni- NTA Superflow Agarose Beads QIAGEN Cat# 30410 Chemical compound, drug Tetrodotoxin Tocris Cat# 1069 Chemical compound, drug Bicuculline methobromide Tocris Cat# 0109 Chemical compound, drug Picrotoxin Sigma- Aldrich Cat# P1675 Chemical compound, drug Okadaic acid Santa Cruz Cat# sc- 3513 Chemical compound, drug Tautomycetin Tocris Cat# 2305 Chemical compound, drug Fostriecin Tocris Cat# 1840 Chemical compound, drug KN62 Tocris Cat# 1277 Chemical compound, drug KN93 Tocris Cat# 1278 Chemical compound, drug H89 Tocris Cat# 2910 Chemical compound, drug Sequencing- grade trypsin Promega Cat# V5111 Chemical compound, drug Tandem Mass Tag (TMT) 10plex Thermo Fisher Scientific Cat# 90110 Software, algorithm Image Lab Software Bio- Rad RRID:SCR_014210 https://www.bio-rad.com/en-us/product/imagelab-software?ID=KRE6P5E8Z&source_wt= imagelabsoftware_surl Software, algorithm ZEN Black Zeiss RRID:SCR_018163 https://www.zeiss.com Software, algorithm Metamorph Molecular Devices RRID:SCR_002368 http://www.moleculardevices.com/Products/Software/ Meta-Imaging-Series/MetaMorph.html Software, algorithm Fiji Fiji RRID:SCR_002285 http://fiji.sc Software, algorithm GraphPad Prism GraphPad RRID:SCR_002798 http://www.graphpad.com/ Software, algorithm IGOR pro Wavemetrics RRID:SCR_000325 https://www.wavemetrics. com/products/igorpro/igorpro.htm Software, algorithm Spectrum mill v.7.00.208 Agilent Technologies Software, algorithm R v 4.0 The R Foundation RRID:SCR_001905 https://www.R-project.org/ Continued Continued on next page Wu, Tatavarty, Jean Beltran, et al. eLife 2022;11:e74277.

Techniques: Activity Assay, Phospho-proteomics, Extraction, Cell Culture, Mass Spectrometry, Western Blot, Functional Assay, Sterility, Comparison, Labeling, Residue, Control

Journal: eLife

Article Title: A bidirectional switch in the Shank3 phosphorylation state biases synapses toward up- or downscaling

doi: 10.7554/elife.74277

Figure Lengend Snippet: Figure 3. Phosphorylation state modulates activity-dependent changes in the synaptic enrichment of Shank3. (A) Representative images of synaptic puncta colocalized with surface GluA2 (sGluA2) and Shank3 in neuron dendrites ± tetrodotoxin (TTX) (scale bar = 5 µm). (B) Quantification of synaptic sGluA2 intensity changes induced by scaling up and down protocols (number of neurons: untreated, n = 77, TTX, n = 40, picrotoxin [PTX], n = 29; Kruskal–Wallis test with post-hoc Dunn’s multiple comparison tests: Un vs. TTX, **p=0.0034, Un vs. PTX, *p=0.0408, TTX vs. PTX, ****p<0.0001). (C) Quantification of synaptic Shank3 intensity during scaling up and down protocols (Kruskal–Wallis test with post-hoc Dunn’s tests: Un vs. TTX, *p=0.0155, Un vs. PTX, *p=0.0205, TTX vs. PTX, ****p<0.0001). (D) Representative images of synaptic localization of wild-type Shank3 and Shank3 phospho-mutants (scale bar = 5 µm). (E) Quantification of synaptic intensity of Shank3 phospho-mutants (number of neurons: WT, n = 33, AA, n = 30, DD, n = 24; Kruskal–Wallis test with post-hoc Dunn’s tests: WT vs. AA, p>0.9999, WT vs. DD, *p=0.0395, AA vs. DD, **p=0.0039). (F) Quantification of the density of synaptic puncta containing Shank3 phospho-mutants (number of neurons: WT, n = 32, AA, n = 30, DD, n = 24; Kruskal–Wallis test: p=0.2814). For imaging experiments here and below, each data point represents a single pyramidal neuron, and data were collected from at least four independent experiments. Also see Figure 3—source data 1.

Article Snippet: DOI: https://doi.org/10.7554/eLife.74277 19 of 31 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Recombinant DNA reagent pAAV- CMV- PI- EGFP- WPRE- bGH Gift from James M. Wilson Addgene# 105530; RRID:Addgene_105530 Commercial assay or kit Lipofectamine 2000 Thermo Fisher Scientific Cat# 11668- 027 Commercial assay or kit Gibson Assembly Master Mix New England Biolabs Cat# E2611S Commercial assay or kit Lambda protein phosphatase New England Biolabs Cat# P0753S Commercial assay or kit BCA Protein Assay Kit Thermo Fisher Scientific Cat# 23227 Commercial assay or kit Protein- G Magnetic Beads Thermo Fisher Scientific Cat# 88847 Commercial assay or kit SimplyBlue SafeStain Thermo Fisher Scientific Cat# LC6060 Commercial assay or kit PP2A Immunoprecipitation Phosphatase Assay Kit Millipore Cat# 17- 313 Commercial assay or kit Ni- NTA Superflow Agarose Beads QIAGEN Cat# 30410 Chemical compound, drug Tetrodotoxin Tocris Cat# 1069 Chemical compound, drug Bicuculline methobromide Tocris Cat# 0109 Chemical compound, drug Picrotoxin Sigma- Aldrich Cat# P1675 Chemical compound, drug Okadaic acid Santa Cruz Cat# sc- 3513 Chemical compound, drug Tautomycetin Tocris Cat# 2305 Chemical compound, drug Fostriecin Tocris Cat# 1840 Chemical compound, drug KN62 Tocris Cat# 1277 Chemical compound, drug KN93 Tocris Cat# 1278 Chemical compound, drug H89 Tocris Cat# 2910 Chemical compound, drug Sequencing- grade trypsin Promega Cat# V5111 Chemical compound, drug Tandem Mass Tag (TMT) 10plex Thermo Fisher Scientific Cat# 90110 Software, algorithm Image Lab Software Bio- Rad RRID:SCR_014210 https://www.bio-rad.com/en-us/product/imagelab-software?ID=KRE6P5E8Z&source_wt= imagelabsoftware_surl Software, algorithm ZEN Black Zeiss RRID:SCR_018163 https://www.zeiss.com Software, algorithm Metamorph Molecular Devices RRID:SCR_002368 http://www.moleculardevices.com/Products/Software/ Meta-Imaging-Series/MetaMorph.html Software, algorithm Fiji Fiji RRID:SCR_002285 http://fiji.sc Software, algorithm GraphPad Prism GraphPad RRID:SCR_002798 http://www.graphpad.com/ Software, algorithm IGOR pro Wavemetrics RRID:SCR_000325 https://www.wavemetrics. com/products/igorpro/igorpro.htm Software, algorithm Spectrum mill v.7.00.208 Agilent Technologies Software, algorithm R v 4.0 The R Foundation RRID:SCR_001905 https://www.R-project.org/ Continued Continued on next page Wu, Tatavarty, Jean Beltran, et al. eLife 2022;11:e74277.

Techniques: Phospho-proteomics, Activity Assay, Comparison, Imaging

Journal: eLife

Article Title: A bidirectional switch in the Shank3 phosphorylation state biases synapses toward up- or downscaling

doi: 10.7554/elife.74277

Figure Lengend Snippet: Figure 4. Increased PP2A activity maintains tetrodotoxin (TTX)-induced Shank3 hypophosphorylation. (A) Diagram showing the potential roles of kinases and phosphatases in regulating activity-dependent Shank3 phosphorylation. (B) Representative Western blot showing the impacts of inhibiting CAMKII (KN62, KN93) or PKA (H89) on Shank3 phosphorylation at baseline and upon TTX treatment. (C) Quantification of S1615 phosphorylation in (B) (two-way ANOVA with post-hoc Tukey’s test: DMSO vs. KN62, p>0.9999, DMSO vs. KN93, p=0.8148, DMSO vs. H89, p=0.9112, DMSO vs. picrotoxin (PTX), *p=0.0406, PTX vs. PTX/KN62, **p=0.0040, PTX vs. PTX/KN93, ****p<0.0001, PTX vs. PTX/H89, ****p<0.0001, n = 5 biological replicates). Dashed line indicates the DMSO control. (D) Quantification of PP2A activity after 1 hr TTX treatment (Un, n = 5, TTX, n = 5; paired t-test: **p=0.0018). (E) Quantification of PP2A activity after 24 hr TTX treatment (Un, n = 7, TTX, n = 7; paired t-test: *p=0.0129). (F, G) Western blot analyses showing changes in S1615 phosphorylation after 1 hr (F) or 24 hr (G) TTX treatment, with inhibition of PP2A by okadaic acid (OKA, 50 nM) during the

Article Snippet: DOI: https://doi.org/10.7554/eLife.74277 19 of 31 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Recombinant DNA reagent pAAV- CMV- PI- EGFP- WPRE- bGH Gift from James M. Wilson Addgene# 105530; RRID:Addgene_105530 Commercial assay or kit Lipofectamine 2000 Thermo Fisher Scientific Cat# 11668- 027 Commercial assay or kit Gibson Assembly Master Mix New England Biolabs Cat# E2611S Commercial assay or kit Lambda protein phosphatase New England Biolabs Cat# P0753S Commercial assay or kit BCA Protein Assay Kit Thermo Fisher Scientific Cat# 23227 Commercial assay or kit Protein- G Magnetic Beads Thermo Fisher Scientific Cat# 88847 Commercial assay or kit SimplyBlue SafeStain Thermo Fisher Scientific Cat# LC6060 Commercial assay or kit PP2A Immunoprecipitation Phosphatase Assay Kit Millipore Cat# 17- 313 Commercial assay or kit Ni- NTA Superflow Agarose Beads QIAGEN Cat# 30410 Chemical compound, drug Tetrodotoxin Tocris Cat# 1069 Chemical compound, drug Bicuculline methobromide Tocris Cat# 0109 Chemical compound, drug Picrotoxin Sigma- Aldrich Cat# P1675 Chemical compound, drug Okadaic acid Santa Cruz Cat# sc- 3513 Chemical compound, drug Tautomycetin Tocris Cat# 2305 Chemical compound, drug Fostriecin Tocris Cat# 1840 Chemical compound, drug KN62 Tocris Cat# 1277 Chemical compound, drug KN93 Tocris Cat# 1278 Chemical compound, drug H89 Tocris Cat# 2910 Chemical compound, drug Sequencing- grade trypsin Promega Cat# V5111 Chemical compound, drug Tandem Mass Tag (TMT) 10plex Thermo Fisher Scientific Cat# 90110 Software, algorithm Image Lab Software Bio- Rad RRID:SCR_014210 https://www.bio-rad.com/en-us/product/imagelab-software?ID=KRE6P5E8Z&source_wt= imagelabsoftware_surl Software, algorithm ZEN Black Zeiss RRID:SCR_018163 https://www.zeiss.com Software, algorithm Metamorph Molecular Devices RRID:SCR_002368 http://www.moleculardevices.com/Products/Software/ Meta-Imaging-Series/MetaMorph.html Software, algorithm Fiji Fiji RRID:SCR_002285 http://fiji.sc Software, algorithm GraphPad Prism GraphPad RRID:SCR_002798 http://www.graphpad.com/ Software, algorithm IGOR pro Wavemetrics RRID:SCR_000325 https://www.wavemetrics. com/products/igorpro/igorpro.htm Software, algorithm Spectrum mill v.7.00.208 Agilent Technologies Software, algorithm R v 4.0 The R Foundation RRID:SCR_001905 https://www.R-project.org/ Continued Continued on next page Wu, Tatavarty, Jean Beltran, et al. eLife 2022;11:e74277.

Techniques: Activity Assay, Phospho-proteomics, Western Blot, Control, Inhibition

Journal: eLife

Article Title: A bidirectional switch in the Shank3 phosphorylation state biases synapses toward up- or downscaling

doi: 10.7554/elife.74277

Figure Lengend Snippet: Figure 5. PP2A activity is required for tetrodotoxin (TTX)-induced synaptic enrichment of Shank3. (A) Representative images of synaptic enrichment of endogenous Shank3 upon treatment with TTX and PP2A inhibitor fostriecin (FST) (scale bar = 10 µm). (B) Quantification of synaptic Shank3 intensity in (A) (number of neurons: DMSO, n = 26, FST, n = 28, TTX, n = 28, TTX/FST, n = 29; Kruskal–Wallis test with post-hoc Dunn’s tests: DMSO vs. FST, p>0.9999, DMSO vs. TTX, ***p=0.0002, FST vs. TTX/FST, p=0.1259, TTX vs. TTX/FST, p=0.1292). (C) Quantification of density of synapses containing Shank3 in (A) (Kruskal–Wallis test with post-hoc Dunn’s tests: DMSO vs. FST, p=0.9458, DMSO vs. TTX, **p=0.0051, FST vs. TTX/FST, p=0.2446, TTX vs. TTX/FST, *p=0.0273). (D) Representative images of synaptic enrichment of endogenous Shank3 upon treatment with TTX and PP1 inhibitor tautomycetin (TAUT) (scale bar = 10 µm). (E) Quantification of synaptic Shank3 intensity in (D) (number of neurons: DMSO, n = 26, TAUT, n = 21, TTX, n = 28, TTX/ TAUT, n = 32; Kruskal–Wallis test with post-hoc Dunn’s tests: DMSO vs. TAUT, *p=0.0315, DMSO vs. TTX, ***p=0.0006, TAUT vs. TTX/TAUT, ***p=0.0002, TTX vs. TTX/TAUT, *p=0.0392). (F) Quantification of density of synapses containing Shank3 in (D) (Kruskal–Wallis test with post-hoc Dunn’s tests: DMSO vs. TAUT, p=0.2450, DMSO vs. TTX, *p=0.0116, TAUT vs. TTX/TAUT, p=0.6552, TTX vs. TTX/TAUT, ***p=0.0007). Also see Figure 5—figure supplement 1 and Figure 5—source data 1.

Article Snippet: DOI: https://doi.org/10.7554/eLife.74277 19 of 31 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Recombinant DNA reagent pAAV- CMV- PI- EGFP- WPRE- bGH Gift from James M. Wilson Addgene# 105530; RRID:Addgene_105530 Commercial assay or kit Lipofectamine 2000 Thermo Fisher Scientific Cat# 11668- 027 Commercial assay or kit Gibson Assembly Master Mix New England Biolabs Cat# E2611S Commercial assay or kit Lambda protein phosphatase New England Biolabs Cat# P0753S Commercial assay or kit BCA Protein Assay Kit Thermo Fisher Scientific Cat# 23227 Commercial assay or kit Protein- G Magnetic Beads Thermo Fisher Scientific Cat# 88847 Commercial assay or kit SimplyBlue SafeStain Thermo Fisher Scientific Cat# LC6060 Commercial assay or kit PP2A Immunoprecipitation Phosphatase Assay Kit Millipore Cat# 17- 313 Commercial assay or kit Ni- NTA Superflow Agarose Beads QIAGEN Cat# 30410 Chemical compound, drug Tetrodotoxin Tocris Cat# 1069 Chemical compound, drug Bicuculline methobromide Tocris Cat# 0109 Chemical compound, drug Picrotoxin Sigma- Aldrich Cat# P1675 Chemical compound, drug Okadaic acid Santa Cruz Cat# sc- 3513 Chemical compound, drug Tautomycetin Tocris Cat# 2305 Chemical compound, drug Fostriecin Tocris Cat# 1840 Chemical compound, drug KN62 Tocris Cat# 1277 Chemical compound, drug KN93 Tocris Cat# 1278 Chemical compound, drug H89 Tocris Cat# 2910 Chemical compound, drug Sequencing- grade trypsin Promega Cat# V5111 Chemical compound, drug Tandem Mass Tag (TMT) 10plex Thermo Fisher Scientific Cat# 90110 Software, algorithm Image Lab Software Bio- Rad RRID:SCR_014210 https://www.bio-rad.com/en-us/product/imagelab-software?ID=KRE6P5E8Z&source_wt= imagelabsoftware_surl Software, algorithm ZEN Black Zeiss RRID:SCR_018163 https://www.zeiss.com Software, algorithm Metamorph Molecular Devices RRID:SCR_002368 http://www.moleculardevices.com/Products/Software/ Meta-Imaging-Series/MetaMorph.html Software, algorithm Fiji Fiji RRID:SCR_002285 http://fiji.sc Software, algorithm GraphPad Prism GraphPad RRID:SCR_002798 http://www.graphpad.com/ Software, algorithm IGOR pro Wavemetrics RRID:SCR_000325 https://www.wavemetrics. com/products/igorpro/igorpro.htm Software, algorithm Spectrum mill v.7.00.208 Agilent Technologies Software, algorithm R v 4.0 The R Foundation RRID:SCR_001905 https://www.R-project.org/ Continued Continued on next page Wu, Tatavarty, Jean Beltran, et al. eLife 2022;11:e74277.

Techniques: Activity Assay

Journal: eLife

Article Title: A bidirectional switch in the Shank3 phosphorylation state biases synapses toward up- or downscaling

doi: 10.7554/elife.74277

Figure Lengend Snippet: Figure 6. Changes in the phosphorylation state of Shank3 are crucial for bidirectional synaptic scaling. (A, B) Representative miniature excitatory postsynaptic current (mEPSC) recordings from neurons overexpressing Shank3 WT (A) or DD mutant (B) during scaling up. (C) Quantification of average mEPSC amplitude in (A) (WT, n = 8, WT + tetrodotoxin [TTX], n = 9; unpaired two-tailed t-test: **p=0.0074). (D) Quantification of average mEPSC amplitude in (B) (number of neurons: DD, n = 12, DD + TTX, n = 14; unpaired two-tailed t-test: p=0.5708). (E, F) Representative traces of mEPSCs recorded from neurons overexpressing Shank3 WT (E) or AA mutant (F) during scaling down. (G) Quantification of average mEPSC amplitude in (E) (number of neurons: WT, n = 8, WT + bicuculline [BIC], n = 8; Mann–Whitney test: *p=0.0148). (H) Quantification of average mEPSC amplitude in (F) (AA, n = 9, AA + BIC, n = 14; unpaired two-tailed t-test: p=0.8612). Also see Figure 6—figure supplement 1, Figure 6—figure supplement 2, and Figure 6—source data 1.

Article Snippet: DOI: https://doi.org/10.7554/eLife.74277 19 of 31 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Recombinant DNA reagent pAAV- CMV- PI- EGFP- WPRE- bGH Gift from James M. Wilson Addgene# 105530; RRID:Addgene_105530 Commercial assay or kit Lipofectamine 2000 Thermo Fisher Scientific Cat# 11668- 027 Commercial assay or kit Gibson Assembly Master Mix New England Biolabs Cat# E2611S Commercial assay or kit Lambda protein phosphatase New England Biolabs Cat# P0753S Commercial assay or kit BCA Protein Assay Kit Thermo Fisher Scientific Cat# 23227 Commercial assay or kit Protein- G Magnetic Beads Thermo Fisher Scientific Cat# 88847 Commercial assay or kit SimplyBlue SafeStain Thermo Fisher Scientific Cat# LC6060 Commercial assay or kit PP2A Immunoprecipitation Phosphatase Assay Kit Millipore Cat# 17- 313 Commercial assay or kit Ni- NTA Superflow Agarose Beads QIAGEN Cat# 30410 Chemical compound, drug Tetrodotoxin Tocris Cat# 1069 Chemical compound, drug Bicuculline methobromide Tocris Cat# 0109 Chemical compound, drug Picrotoxin Sigma- Aldrich Cat# P1675 Chemical compound, drug Okadaic acid Santa Cruz Cat# sc- 3513 Chemical compound, drug Tautomycetin Tocris Cat# 2305 Chemical compound, drug Fostriecin Tocris Cat# 1840 Chemical compound, drug KN62 Tocris Cat# 1277 Chemical compound, drug KN93 Tocris Cat# 1278 Chemical compound, drug H89 Tocris Cat# 2910 Chemical compound, drug Sequencing- grade trypsin Promega Cat# V5111 Chemical compound, drug Tandem Mass Tag (TMT) 10plex Thermo Fisher Scientific Cat# 90110 Software, algorithm Image Lab Software Bio- Rad RRID:SCR_014210 https://www.bio-rad.com/en-us/product/imagelab-software?ID=KRE6P5E8Z&source_wt= imagelabsoftware_surl Software, algorithm ZEN Black Zeiss RRID:SCR_018163 https://www.zeiss.com Software, algorithm Metamorph Molecular Devices RRID:SCR_002368 http://www.moleculardevices.com/Products/Software/ Meta-Imaging-Series/MetaMorph.html Software, algorithm Fiji Fiji RRID:SCR_002285 http://fiji.sc Software, algorithm GraphPad Prism GraphPad RRID:SCR_002798 http://www.graphpad.com/ Software, algorithm IGOR pro Wavemetrics RRID:SCR_000325 https://www.wavemetrics. com/products/igorpro/igorpro.htm Software, algorithm Spectrum mill v.7.00.208 Agilent Technologies Software, algorithm R v 4.0 The R Foundation RRID:SCR_001905 https://www.R-project.org/ Continued Continued on next page Wu, Tatavarty, Jean Beltran, et al. eLife 2022;11:e74277.

Techniques: Phospho-proteomics, Mutagenesis, Two Tailed Test, MANN-WHITNEY

Journal: eLife

Article Title: A bidirectional switch in the Shank3 phosphorylation state biases synapses toward up- or downscaling

doi: 10.7554/elife.74277

Figure Lengend Snippet: Figure 7. Brief PP2A inactivation reverses scaling up. (A) Representative images showing the effects of 1 hr fostriecin (FST) treatment on synaptic sGluA2 intensity in neurons expressing Shank3 WT or AA, after 24 hr of tetrodotoxin (TTX) to scale up synaptic strengths (scale bar = 10 µm). (B) Quantification of synaptic sGluA2 intensity in (A) (number of cells: WT/TTX, n = 22, WT/TTX/FST, n = 23, AA/TTX, n = 26, AA/TTX/FST, n = 25; Mann–Whitney test: WT/TTX vs. WT/TTX/FST, ***p=0.0007, AA/TTX vs. AA/TTX/FST, p=0.3739). (C) Quantification of synaptic Shank3 intensity in (A) (Mann–Whitney test: WT/TTX vs. WT/TTX/FST, **p=0.0090, AA/TTX vs. AA/TTX/FST, p=0.7296). (D) Quantification of the density of puncta containing sGluA2 and Shank3 (Mann–Whitney test: WT/TTX vs. WT/TTX/FST, **p=0.0016, AA/TTX vs. AA/TTX/FST, p=0.7017). Each data point indicates a cell, and the total number (n) was pooled from five independent experiments. Also see Figure 7—source data 1.

Article Snippet: DOI: https://doi.org/10.7554/eLife.74277 19 of 31 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Recombinant DNA reagent pAAV- CMV- PI- EGFP- WPRE- bGH Gift from James M. Wilson Addgene# 105530; RRID:Addgene_105530 Commercial assay or kit Lipofectamine 2000 Thermo Fisher Scientific Cat# 11668- 027 Commercial assay or kit Gibson Assembly Master Mix New England Biolabs Cat# E2611S Commercial assay or kit Lambda protein phosphatase New England Biolabs Cat# P0753S Commercial assay or kit BCA Protein Assay Kit Thermo Fisher Scientific Cat# 23227 Commercial assay or kit Protein- G Magnetic Beads Thermo Fisher Scientific Cat# 88847 Commercial assay or kit SimplyBlue SafeStain Thermo Fisher Scientific Cat# LC6060 Commercial assay or kit PP2A Immunoprecipitation Phosphatase Assay Kit Millipore Cat# 17- 313 Commercial assay or kit Ni- NTA Superflow Agarose Beads QIAGEN Cat# 30410 Chemical compound, drug Tetrodotoxin Tocris Cat# 1069 Chemical compound, drug Bicuculline methobromide Tocris Cat# 0109 Chemical compound, drug Picrotoxin Sigma- Aldrich Cat# P1675 Chemical compound, drug Okadaic acid Santa Cruz Cat# sc- 3513 Chemical compound, drug Tautomycetin Tocris Cat# 2305 Chemical compound, drug Fostriecin Tocris Cat# 1840 Chemical compound, drug KN62 Tocris Cat# 1277 Chemical compound, drug KN93 Tocris Cat# 1278 Chemical compound, drug H89 Tocris Cat# 2910 Chemical compound, drug Sequencing- grade trypsin Promega Cat# V5111 Chemical compound, drug Tandem Mass Tag (TMT) 10plex Thermo Fisher Scientific Cat# 90110 Software, algorithm Image Lab Software Bio- Rad RRID:SCR_014210 https://www.bio-rad.com/en-us/product/imagelab-software?ID=KRE6P5E8Z&source_wt= imagelabsoftware_surl Software, algorithm ZEN Black Zeiss RRID:SCR_018163 https://www.zeiss.com Software, algorithm Metamorph Molecular Devices RRID:SCR_002368 http://www.moleculardevices.com/Products/Software/ Meta-Imaging-Series/MetaMorph.html Software, algorithm Fiji Fiji RRID:SCR_002285 http://fiji.sc Software, algorithm GraphPad Prism GraphPad RRID:SCR_002798 http://www.graphpad.com/ Software, algorithm IGOR pro Wavemetrics RRID:SCR_000325 https://www.wavemetrics. com/products/igorpro/igorpro.htm Software, algorithm Spectrum mill v.7.00.208 Agilent Technologies Software, algorithm R v 4.0 The R Foundation RRID:SCR_001905 https://www.R-project.org/ Continued Continued on next page Wu, Tatavarty, Jean Beltran, et al. eLife 2022;11:e74277.

Techniques: Expressing, MANN-WHITNEY

Journal: Nucleic Acids Research

Article Title: A cancer persistent DNA repair circuit driven by MDM2, MDM4 (MDMX), and mutant p53 for recruitment of MDC1 and 53BP1 on chromatin

doi: 10.1093/nar/gkaf627

Figure Lengend Snippet: Reduced MDM2 protein in T47D cells causes reduced chromatin phosphoproteins 53BP1 and MDC1. ( A ) Experimental design workflow of the SILAC analysis (created in BioRender. Harmon, K. (2025) https://BioRender.com/oyd7ala ). Chromatin isolated from a mixture of T47D vector control cells (MDM2-competent) cultured in natural amino acid medium, and T47Dshmdm2 cells (MDM2-depleted) cultured in heavy isotope amino acid medium, was subjected to proteolysis followed by phospho-peptide purification and enrichment. Scatter plot represents the H/L ratio versus abundance of peptides identified by mass spectrometry, with those corresponding to TP53BP1 (magenta), TP53 (blue), MCM2 (green), and MDC1(brown) highlighted. ( B ) Chromatin (5 μg) isolated from T47D vector control, T47Dshmdm2, and T47Dshmdmx cells was subjected to SDS–PAGE/western blot analysis for 53BP1, MDC1, MCM4, lamin A/C, and mtp53. ( C and D ) IF of total 53BP1 (i), phospho-53BP1 ser25 (ii), or phospho- 53BP1 ser1778 (iii) within T47D vector control nuclei [(i) 528, (ii) 549, and (iii) 501], T47Dshmdm2 nuclei [(i) 549,(ii) 520, and (iii) 501], and T47Dshmdmx nuclei [(i) 623, (ii) 376, and (iii) 501]. Confocal images for six fields for each were acquired and the number of 53BP1 foci per nucleus from each cell population indicated above was determined. Representative data ( n = 3) with mean, 95% CI, and Kruskal–Wallis statistical significance test prepared as described in the “Materials and methods” section. **** Indicates a p-value less than or equal to 0.0001, ** indicates a p-value less than or equal to 0.01, * indicates a p-value less than or equal to 0.05, and ns is nonsignificant.

Article Snippet: Antibodies used for western blotting (WB), immunofluorescence staining (IF), immunoprecipitation (IP), and proximity ligation assay (PLA) were purchased from the following (usage denoted in parenthesis): rabbit p53 Sigma cat# A300-247A (PLA), and Proteintech cat# 10442-1- AP (WB); mouse p53 DO1 Santa Cruz Biotechnology cat# sc-126 (PLA and WB); mouse p53 DO1-HRP Santa Cruz Biotechnology cat# sc-126 HRP (WB); rabbit MDMX Proteintech cat# 17914-1-AP (WB); [ ] rabbit MDM2 R&D Systems cat# AF1244 (WB); rabbit 53BP1 Cell Signaling Technology cat# 4937 (WB and IF); rabbit phospho-Serine 177853BP1 Cell Signaling Technology cat# 2675 (WB and IF); rabbit phospho-Serine 2553BP1 Sigma cat# PLA 0126 (WB, IF, and PLA); rabbit MDC1 Sigma cat# PLA0016 (WB, IF, and PLA); rabbit MCM4 Cell Signaling Technology cat# 12973 (WB); mouse Actin-HRP Sigma cat# A3854 (WB); [ ] mouse Lamin A cat# SAB4200420 (WB); mouse PARP1 BD Biosciences cat# 51-6639GR (WB); goat 53BP1 Sigma cat# PLA0303 (PLA and IP); goat anti-mouse HRP Sigma cat# A3682 (WB); goat anti-rabbit Proteintech cat# SA00001-2 (WB); mouse Cyclin A Santa Cruz Biotechnology cat# sc-271682 (WB); rabbit Cyclin A Cell Signaling Technology cat# 67955S (IF); mouse Cyclin B Santa Cruz Biotechnology cat# sc-245 (WB); rabbit p21 Cell Signaling Technology cat# 2947S (WB); γH2AX phospho-Ser139 Cell Signaling Technology cat# 9718S (WB and IF); rabbit Poly ADP-Ribose Cell Signaling Technology cat# 83732S (WB); mouse MDM2 SMP14 Santa Cruz Biotechnology cat# sc-965 (IP); mouse IgG Santa Cruz Biotechnology cat# sc-2025 (IP); Purified mouse MDM2 4B2 [ ]; and purified mouse MDM2 2A9 [ ] were used for PLA and IP and prepared as described [ ].

Techniques: Multiplex sample analysis, Isolation, Plasmid Preparation, Control, Cell Culture, Purification, Mass Spectrometry, SDS Page, Western Blot

Journal: Nucleic Acids Research

Article Title: A cancer persistent DNA repair circuit driven by MDM2, MDM4 (MDMX), and mutant p53 for recruitment of MDC1 and 53BP1 on chromatin

doi: 10.1093/nar/gkaf627

Figure Lengend Snippet: 53BP1 in breast cancer cells interacts with both mtp53 and MDM2. ( A ) Relative abundance of mtp53, MDM2, MDMX, 53BP1, and MDC1 within whole cell extracts (20 μg) prepared from T47D (L194F) and MDA-MB-231 (R280K) cell lines determined by SDS–PAGE/western blot analysis. ( B–D ) Association of mtp53, MDM2, and 53BP1 in vivo measured using the PLA. PLA analyses of mtp53-53BP1 (panel B), MDM2-mtp53 (panel C), and MDM2-53BP1 (panel D) within T47D and MDA-MB-231 cells were performed as described in the “Materials and methods” section; primary antibodies are PLA rabbit anti-p53, PLA goat anti-53BP1, and mouse anti-MDM2 4B2. Confocal images for 4–6 fields for each were acquired and the number of PLA foci per nucleus for each cell population was determined ( n = 3 for T47D, n = 2 for MDA-MB-231). Representative data with mean, 95% CI, and Kruskal–Wallis statistical significance test prepared as described in the “Materials and methods” section from the indicated number of cells: Panel B vector (T47D = 276; MDA-MB-231 = 108), shmdmx (T47D = 239; MDA-MB-231 = 128), and shmdm2 (T47D = 310; MDA-MB-231 = 106); Panel C vector (T47D = 92; MDA-MB-231 = 104), shmdmx (T47D = 108; MDA-MB-231 = 131), and shmdm2 (T47D = 101; MDA-MB-231 = 136); Panel D vector (T47D = 761; MDA-MB-231 = 129), shmdmx (T47D = 529; MDA-MB-231 = 142), and shmdm2 (T47D = 964; MDA-MB-231 = 163). **** Indicates a p-value less than or equal to 0.0001, ** indicates a p-value less than or equal to 0.01, * indicates a p-value less than or equal to 0.05, and ns is nonsignificant.

Article Snippet: Antibodies used for western blotting (WB), immunofluorescence staining (IF), immunoprecipitation (IP), and proximity ligation assay (PLA) were purchased from the following (usage denoted in parenthesis): rabbit p53 Sigma cat# A300-247A (PLA), and Proteintech cat# 10442-1- AP (WB); mouse p53 DO1 Santa Cruz Biotechnology cat# sc-126 (PLA and WB); mouse p53 DO1-HRP Santa Cruz Biotechnology cat# sc-126 HRP (WB); rabbit MDMX Proteintech cat# 17914-1-AP (WB); [ ] rabbit MDM2 R&D Systems cat# AF1244 (WB); rabbit 53BP1 Cell Signaling Technology cat# 4937 (WB and IF); rabbit phospho-Serine 177853BP1 Cell Signaling Technology cat# 2675 (WB and IF); rabbit phospho-Serine 2553BP1 Sigma cat# PLA 0126 (WB, IF, and PLA); rabbit MDC1 Sigma cat# PLA0016 (WB, IF, and PLA); rabbit MCM4 Cell Signaling Technology cat# 12973 (WB); mouse Actin-HRP Sigma cat# A3854 (WB); [ ] mouse Lamin A cat# SAB4200420 (WB); mouse PARP1 BD Biosciences cat# 51-6639GR (WB); goat 53BP1 Sigma cat# PLA0303 (PLA and IP); goat anti-mouse HRP Sigma cat# A3682 (WB); goat anti-rabbit Proteintech cat# SA00001-2 (WB); mouse Cyclin A Santa Cruz Biotechnology cat# sc-271682 (WB); rabbit Cyclin A Cell Signaling Technology cat# 67955S (IF); mouse Cyclin B Santa Cruz Biotechnology cat# sc-245 (WB); rabbit p21 Cell Signaling Technology cat# 2947S (WB); γH2AX phospho-Ser139 Cell Signaling Technology cat# 9718S (WB and IF); rabbit Poly ADP-Ribose Cell Signaling Technology cat# 83732S (WB); mouse MDM2 SMP14 Santa Cruz Biotechnology cat# sc-965 (IP); mouse IgG Santa Cruz Biotechnology cat# sc-2025 (IP); Purified mouse MDM2 4B2 [ ]; and purified mouse MDM2 2A9 [ ] were used for PLA and IP and prepared as described [ ].

Techniques: SDS Page, Western Blot, In Vivo, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: A cancer persistent DNA repair circuit driven by MDM2, MDM4 (MDMX), and mutant p53 for recruitment of MDC1 and 53BP1 on chromatin

doi: 10.1093/nar/gkaf627

Figure Lengend Snippet: The MDM2–53BP1 interaction is promoted by the mtp53 C-terminus. ( A ) The C-terminus of mtp53 R273H within MDA-MB-468 was modified using CRISPR–Cas9 to create the cell line MDA-MB-468 R273Hfs347Δ360-393 (termed G6; mtp53 derivative R273HΔC). ( B ) Relative protein levels of 53BP1, MDM2, and mtp53 within MDA-MB-468 (25, 12.5, 6.25, and 3.125 μg) and G6 (25 μg) cell lines was examined by SDS–PAGE/western blot analysis. ( C ) Loss of mtp53 C-terminus disrupts mtp53 co-IP with MDM2. Extracts from MDA-MB-468 and G6 cell lines were incubated with either normal mouse IgG (negative control) or anti-MDM2 antibodies 4B2 (lanes 1–7) or SMP14 (lanes 8–14), and IP reactions were examined for MDM2 and mtp53 by western blot analysis. Lanes 1–7: 4B2 IP reactions from 800 μg of extract (input); lanes contain 10% of total IP and 2% of input. Lanes 8–14: SMP14 IP reactions from 1600 μg of extract; lanes contain 12.5% each IP and 0.5% of input. Lanes labeled 2× (lane 7 for the 4B2 IPs and lane 14 for the SMP14 IPs) contain twice the amount of the G6 extract MDM2 IP; a lighter exposure of mtp53 input is presented due to the vast excess of mtp53 compared to MDM2 within the MDA-MB-468 cell lines. ( D–F ) PLA analysis of mtp53–53BP1 (panel D), MDM2–mtp53 (panel E), and MDM2–53BP1 (panel F) in MDA-MB-468 and G6. PLA analysis of the indicated proteins was measured using PLA rabbit anti-p53, PLA goat anti-53BP1, and mouse anti-MDM2 2A9 antibodies. Confocal images for 3–5 fields for each were acquired and the number of PLA foci per nucleus for each cell population was determined ( n = 2). Representative data with mean, 95% CI, and Kruskal–Wallis statistical significance test prepared as described in the “Materials and methods” section from the indicated number of cells in panel: (D) R273H = 120; R273HΔC = 144; (E) R273H = 131; R273HΔC = 159; (F) R273H = 102; R273HΔC = 138. **** Indicates a p-value less than or equal to 0.0001.

Article Snippet: Antibodies used for western blotting (WB), immunofluorescence staining (IF), immunoprecipitation (IP), and proximity ligation assay (PLA) were purchased from the following (usage denoted in parenthesis): rabbit p53 Sigma cat# A300-247A (PLA), and Proteintech cat# 10442-1- AP (WB); mouse p53 DO1 Santa Cruz Biotechnology cat# sc-126 (PLA and WB); mouse p53 DO1-HRP Santa Cruz Biotechnology cat# sc-126 HRP (WB); rabbit MDMX Proteintech cat# 17914-1-AP (WB); [ ] rabbit MDM2 R&D Systems cat# AF1244 (WB); rabbit 53BP1 Cell Signaling Technology cat# 4937 (WB and IF); rabbit phospho-Serine 177853BP1 Cell Signaling Technology cat# 2675 (WB and IF); rabbit phospho-Serine 2553BP1 Sigma cat# PLA 0126 (WB, IF, and PLA); rabbit MDC1 Sigma cat# PLA0016 (WB, IF, and PLA); rabbit MCM4 Cell Signaling Technology cat# 12973 (WB); mouse Actin-HRP Sigma cat# A3854 (WB); [ ] mouse Lamin A cat# SAB4200420 (WB); mouse PARP1 BD Biosciences cat# 51-6639GR (WB); goat 53BP1 Sigma cat# PLA0303 (PLA and IP); goat anti-mouse HRP Sigma cat# A3682 (WB); goat anti-rabbit Proteintech cat# SA00001-2 (WB); mouse Cyclin A Santa Cruz Biotechnology cat# sc-271682 (WB); rabbit Cyclin A Cell Signaling Technology cat# 67955S (IF); mouse Cyclin B Santa Cruz Biotechnology cat# sc-245 (WB); rabbit p21 Cell Signaling Technology cat# 2947S (WB); γH2AX phospho-Ser139 Cell Signaling Technology cat# 9718S (WB and IF); rabbit Poly ADP-Ribose Cell Signaling Technology cat# 83732S (WB); mouse MDM2 SMP14 Santa Cruz Biotechnology cat# sc-965 (IP); mouse IgG Santa Cruz Biotechnology cat# sc-2025 (IP); Purified mouse MDM2 4B2 [ ]; and purified mouse MDM2 2A9 [ ] were used for PLA and IP and prepared as described [ ].

Techniques: Modification, CRISPR, SDS Page, Western Blot, Co-Immunoprecipitation Assay, Incubation, Negative Control, Labeling

Journal: Nucleic Acids Research

Article Title: A cancer persistent DNA repair circuit driven by MDM2, MDM4 (MDMX), and mutant p53 for recruitment of MDC1 and 53BP1 on chromatin

doi: 10.1093/nar/gkaf627

Figure Lengend Snippet: The MDM2-mtp53 and MDM2–53BP1 interactions are Nutlin 3a sensitive. ( A ) Western blot analysis of whole cell extracts (10 μg) from MCF7 and T47D cell line populations treated for the indicated time with either vehicle or 10 μM Nutlin 3a (labeled N3a or “+” in graphs) for the indicated proteins. ( B ) Nutlin 3a does not inhibit T47D cell cycle progression. Twenty-four hour post-treatment with either vehicle or 10 μM Nutlin 3a, cells were labeled with EdU for 20 min and assayed for Cyclin A2 by immunofluorescence. Confocal images from at least three fields were acquired and the number of EdU + and Cyclin A + nuclei were quantified (tabulated in S4, panel C) in each population of vehicle-treated [vector = 289; shmdmx = 278; shmdm2 = 249] and Nutlin 3a-treated [vector = 227; shmdmx = 283; shmdm2 = 245] cells. The S/G2 fraction (total Cyclin A + nuclei) for vehicle-treated: vector = 33.6%, shmdmx = 37.8%, shmdm2 = 36.8%; Nutlin 3a-treated: vector = 38.3%, shmdmx = 27.0%, shmdm2 = 29.4%. ( C and D ) Nutlin 3a disrupts mtp53–MDM2 and 53BP1–MDM2 PLA foci in T47D. Twenty-four hour post-treatment with vehicle or 10 μM Nutlin 3a cell populations PLA interactions were measured between MDM2–mtp53 (panel C) and MDM2–53BP1 (panel D) using PLA rabbit anti-p53, PLA goat anti-53BP1, and mouse anti-MDM2 4B2 primary antibodies. Confocal images for 4–6 fields for each were acquired and the number of PLA foci per nucleus from each cell population was determined ( n = 2 for panel C and n = 3 for panel D). Representative data with mean, 95% CI, and Kruskal–Wallis statistical significance test prepared as described in the materials and methods from the indicated number of cells in panel: (C) vehicle-treated [vector = 328; shmdmx = 298; shmdm2 = 249], Nutlin 3a-treated [vector = 323; shmdmx = 364; shmdm2 = 285] and (D) vehicle-treated [vector = 543; shmdmx = 548; shmdm2 = 455], Nutlin 3a-treated [vector = 610; shmdmx = 545; shmdm2 = 618]. **** Indicates a p-value less than or equal to 0.0001.

Article Snippet: Antibodies used for western blotting (WB), immunofluorescence staining (IF), immunoprecipitation (IP), and proximity ligation assay (PLA) were purchased from the following (usage denoted in parenthesis): rabbit p53 Sigma cat# A300-247A (PLA), and Proteintech cat# 10442-1- AP (WB); mouse p53 DO1 Santa Cruz Biotechnology cat# sc-126 (PLA and WB); mouse p53 DO1-HRP Santa Cruz Biotechnology cat# sc-126 HRP (WB); rabbit MDMX Proteintech cat# 17914-1-AP (WB); [ ] rabbit MDM2 R&D Systems cat# AF1244 (WB); rabbit 53BP1 Cell Signaling Technology cat# 4937 (WB and IF); rabbit phospho-Serine 177853BP1 Cell Signaling Technology cat# 2675 (WB and IF); rabbit phospho-Serine 2553BP1 Sigma cat# PLA 0126 (WB, IF, and PLA); rabbit MDC1 Sigma cat# PLA0016 (WB, IF, and PLA); rabbit MCM4 Cell Signaling Technology cat# 12973 (WB); mouse Actin-HRP Sigma cat# A3854 (WB); [ ] mouse Lamin A cat# SAB4200420 (WB); mouse PARP1 BD Biosciences cat# 51-6639GR (WB); goat 53BP1 Sigma cat# PLA0303 (PLA and IP); goat anti-mouse HRP Sigma cat# A3682 (WB); goat anti-rabbit Proteintech cat# SA00001-2 (WB); mouse Cyclin A Santa Cruz Biotechnology cat# sc-271682 (WB); rabbit Cyclin A Cell Signaling Technology cat# 67955S (IF); mouse Cyclin B Santa Cruz Biotechnology cat# sc-245 (WB); rabbit p21 Cell Signaling Technology cat# 2947S (WB); γH2AX phospho-Ser139 Cell Signaling Technology cat# 9718S (WB and IF); rabbit Poly ADP-Ribose Cell Signaling Technology cat# 83732S (WB); mouse MDM2 SMP14 Santa Cruz Biotechnology cat# sc-965 (IP); mouse IgG Santa Cruz Biotechnology cat# sc-2025 (IP); Purified mouse MDM2 4B2 [ ]; and purified mouse MDM2 2A9 [ ] were used for PLA and IP and prepared as described [ ].

Techniques: Western Blot, Labeling, Immunofluorescence, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: A cancer persistent DNA repair circuit driven by MDM2, MDM4 (MDMX), and mutant p53 for recruitment of MDC1 and 53BP1 on chromatin

doi: 10.1093/nar/gkaf627

Figure Lengend Snippet: MDM2 promotes 53BP1–MDC1 complex formation. ( A ) Reduced 53BP1–MDC1 PLA foci in T47D lacking MDM2. EdU-labeled T47D vector and shmdm2 cells were assayed for 53BP1–MDC1 PLA foci using PLA goat anti-53BP1 and PLA rabbit anti-MDC1 antibodies. Shown under representative images ( n = 3) is the number of EdU + nuclei identified within each cell population and graphed is the number of PLA foci per nucleus. For the PLA analysis representative data with mean, 95% CI, and Kruskal–Wallis statistical significance test prepared as described in the “Materials and methods” section from the indicated number of cells: vector (total = 329, S-phase = 100, G1-G2 = 229); shmdm2 (total = 251, S-phase = 79, G1-G2 = 172). ( B ) Activation of the DDR in T47D cell lines by Etoposide but not Nutlin-3a. Whole cell extracts (20 μg) from T47D cell lines treated with either 50 μM Etoposide for 5 h (Etop), or 10 μM Nutlin 3a for 24 h (N3a) were analyzed for the indicated proteins by WB. ( C and D ) MDC1–53BP1 PLA foci are disrupted by both DDR activation and Nutlin 3a. MDC1–53BP1 PLA analyses were performed within each T47D cell line at the indicated time points post Etoposide treatment (panel C) or 24 h-post Nutlin 3a treatment (panel D) using PLA goat anti-53BP1 and PLA rabbit anti-MDC1. Confocal images for 3–6 fields for each were acquired and the number of PLA per nucleus from each cell population was determined ( n = 3 for panel C and n = 3 for panel D). Representative data with mean, 95% CI, and Kruskal–Wallis statistical significance test prepared as described in the “Materials and methods” section from the indicated number of cells in panel: (C) vehicle-treated (0 h Etop) [vector = 208; shmdmx = 198; shmdm2 = 247], 2 h Etoposide [vector = 205; shmdmx = 184; shmdm2 = 231], 5 h Etoposide [vector = 189; shmdmx = 224; shmdm2 = 176]; (D) vehicle-treated [vector = 198; shmdmx = 270; shmdm2 = 336] and Nutlin 3a-treated [vector = 380; shmdmx = 339; shmdm2 = 252]. **** Indicates a p-value less than or equal to 0.0001, ** indicates a p-value less than or equal to 0.01, * indicates a p-value less than or equal to 0.05, and ns is nonsignificant.

Article Snippet: Antibodies used for western blotting (WB), immunofluorescence staining (IF), immunoprecipitation (IP), and proximity ligation assay (PLA) were purchased from the following (usage denoted in parenthesis): rabbit p53 Sigma cat# A300-247A (PLA), and Proteintech cat# 10442-1- AP (WB); mouse p53 DO1 Santa Cruz Biotechnology cat# sc-126 (PLA and WB); mouse p53 DO1-HRP Santa Cruz Biotechnology cat# sc-126 HRP (WB); rabbit MDMX Proteintech cat# 17914-1-AP (WB); [ ] rabbit MDM2 R&D Systems cat# AF1244 (WB); rabbit 53BP1 Cell Signaling Technology cat# 4937 (WB and IF); rabbit phospho-Serine 177853BP1 Cell Signaling Technology cat# 2675 (WB and IF); rabbit phospho-Serine 2553BP1 Sigma cat# PLA 0126 (WB, IF, and PLA); rabbit MDC1 Sigma cat# PLA0016 (WB, IF, and PLA); rabbit MCM4 Cell Signaling Technology cat# 12973 (WB); mouse Actin-HRP Sigma cat# A3854 (WB); [ ] mouse Lamin A cat# SAB4200420 (WB); mouse PARP1 BD Biosciences cat# 51-6639GR (WB); goat 53BP1 Sigma cat# PLA0303 (PLA and IP); goat anti-mouse HRP Sigma cat# A3682 (WB); goat anti-rabbit Proteintech cat# SA00001-2 (WB); mouse Cyclin A Santa Cruz Biotechnology cat# sc-271682 (WB); rabbit Cyclin A Cell Signaling Technology cat# 67955S (IF); mouse Cyclin B Santa Cruz Biotechnology cat# sc-245 (WB); rabbit p21 Cell Signaling Technology cat# 2947S (WB); γH2AX phospho-Ser139 Cell Signaling Technology cat# 9718S (WB and IF); rabbit Poly ADP-Ribose Cell Signaling Technology cat# 83732S (WB); mouse MDM2 SMP14 Santa Cruz Biotechnology cat# sc-965 (IP); mouse IgG Santa Cruz Biotechnology cat# sc-2025 (IP); Purified mouse MDM2 4B2 [ ]; and purified mouse MDM2 2A9 [ ] were used for PLA and IP and prepared as described [ ].

Techniques: Labeling, Plasmid Preparation, Activation Assay

Journal: Nucleic Acids Research

Article Title: A cancer persistent DNA repair circuit driven by MDM2, MDM4 (MDMX), and mutant p53 for recruitment of MDC1 and 53BP1 on chromatin

doi: 10.1093/nar/gkaf627

Figure Lengend Snippet: Co-IP demonstrates a 53BP1–MDC1–MDM2 multiprotein complex. ( A ) Co-IP demonstrates a 53BP1–MDC1–MDM2 multiprotein complex. 53BP1 was immunoprecipitated from T47D CES as described in the “Materials and methods” section subjected to western blot analysis for 53BP1, MDC1, MDM2, and p53. Lanes are as follows: (1) cytoplasmic extract, (2) CES IP input, (3) IP with IgG, and (4) IP for 53BP1. ( B ) Inhibition of ATM promotes MDM2 activity. Western blot analysis for the indicated proteins within extracts (10 μg) from T47D populations treated for 24 h with either vehicle,10 μM ALRN-6924 (MDM2/X dual inhibitor), 10 μM KU-55933 (ATMi; ATM inhibitor) or both. ( C ) The MDM2 inhibitor ALRN-6924 reduces whereas the ATM inhibitor increases MDC1–53BP1 PLA foci in T47D. Twenty-four hour post treatment with either vehicle,10 μM ALRN-6924, 10 μM KU-55933 (ATMi) or both T47D vector and shmdm2 cells were labeled with EdU for 20 min and then assayed for 53BP1–MDC1 PLA foci using PLA goat anti-53BP1 and PLA rabbit anti-MDC1 antibodies. Confocal images for several fields were acquired and the number of PLA foci/nucleus from each cell population was determined. Representative data with mean, 95% CI, and Kruskal–Wallis statistical significance test prepared as described in the “Materials and methods” section from the indicated number of cells in: vehicle-treated [vector = 186; shmdm2 = 87], ATMi-treated [vector = 163; shmdm2 = 188], ALRN-6924-treated [vector = 196; shmdm2 = 176], ALRN-6924 + ATMi-treated [vector = 154; shmdm2 = 159]. **** Indicates a p-value less than or equal to 0.0001, ** indicates a p-value less than or equal to 0.01, * indicates a p-value less than or equal to 0.05, and ns is nonsignificant.

Article Snippet: Antibodies used for western blotting (WB), immunofluorescence staining (IF), immunoprecipitation (IP), and proximity ligation assay (PLA) were purchased from the following (usage denoted in parenthesis): rabbit p53 Sigma cat# A300-247A (PLA), and Proteintech cat# 10442-1- AP (WB); mouse p53 DO1 Santa Cruz Biotechnology cat# sc-126 (PLA and WB); mouse p53 DO1-HRP Santa Cruz Biotechnology cat# sc-126 HRP (WB); rabbit MDMX Proteintech cat# 17914-1-AP (WB); [ ] rabbit MDM2 R&D Systems cat# AF1244 (WB); rabbit 53BP1 Cell Signaling Technology cat# 4937 (WB and IF); rabbit phospho-Serine 177853BP1 Cell Signaling Technology cat# 2675 (WB and IF); rabbit phospho-Serine 2553BP1 Sigma cat# PLA 0126 (WB, IF, and PLA); rabbit MDC1 Sigma cat# PLA0016 (WB, IF, and PLA); rabbit MCM4 Cell Signaling Technology cat# 12973 (WB); mouse Actin-HRP Sigma cat# A3854 (WB); [ ] mouse Lamin A cat# SAB4200420 (WB); mouse PARP1 BD Biosciences cat# 51-6639GR (WB); goat 53BP1 Sigma cat# PLA0303 (PLA and IP); goat anti-mouse HRP Sigma cat# A3682 (WB); goat anti-rabbit Proteintech cat# SA00001-2 (WB); mouse Cyclin A Santa Cruz Biotechnology cat# sc-271682 (WB); rabbit Cyclin A Cell Signaling Technology cat# 67955S (IF); mouse Cyclin B Santa Cruz Biotechnology cat# sc-245 (WB); rabbit p21 Cell Signaling Technology cat# 2947S (WB); γH2AX phospho-Ser139 Cell Signaling Technology cat# 9718S (WB and IF); rabbit Poly ADP-Ribose Cell Signaling Technology cat# 83732S (WB); mouse MDM2 SMP14 Santa Cruz Biotechnology cat# sc-965 (IP); mouse IgG Santa Cruz Biotechnology cat# sc-2025 (IP); Purified mouse MDM2 4B2 [ ]; and purified mouse MDM2 2A9 [ ] were used for PLA and IP and prepared as described [ ].

Techniques: Co-Immunoprecipitation Assay, Immunoprecipitation, Western Blot, Inhibition, Activity Assay, Plasmid Preparation, Labeling

Journal: Nucleic Acids Research

Article Title: A cancer persistent DNA repair circuit driven by MDM2, MDM4 (MDMX), and mutant p53 for recruitment of MDC1 and 53BP1 on chromatin

doi: 10.1093/nar/gkaf627

Figure Lengend Snippet: Depletion of MDM2 increases poly(ADP-ribose) modification (PARylation) levels of chromatin bound proteins. Cytosolic ( A ) and chromatin ( B ) fractions were prepared from T47D cells with constitutive shmdm2, shmdmx, or mir30 shRNA-expressing vector cells treated with either vehicle (DMSO), or a combination of 1 mM temozolomide plus10 μM talazoparib (Temo + Tal) for 4 h, or combination of 1 mM temozolomide plus10 μM talazoparib (Temo + Tal) for 4 h and then replaced with fresh media for an additional 24 h. Ten micrograms of cytosolic or chromatin protein was loaded on a SDS–PAGE and protein levels were determined by western blot analysis using the indicated antibodies.

Article Snippet: Antibodies used for western blotting (WB), immunofluorescence staining (IF), immunoprecipitation (IP), and proximity ligation assay (PLA) were purchased from the following (usage denoted in parenthesis): rabbit p53 Sigma cat# A300-247A (PLA), and Proteintech cat# 10442-1- AP (WB); mouse p53 DO1 Santa Cruz Biotechnology cat# sc-126 (PLA and WB); mouse p53 DO1-HRP Santa Cruz Biotechnology cat# sc-126 HRP (WB); rabbit MDMX Proteintech cat# 17914-1-AP (WB); [ ] rabbit MDM2 R&D Systems cat# AF1244 (WB); rabbit 53BP1 Cell Signaling Technology cat# 4937 (WB and IF); rabbit phospho-Serine 177853BP1 Cell Signaling Technology cat# 2675 (WB and IF); rabbit phospho-Serine 2553BP1 Sigma cat# PLA 0126 (WB, IF, and PLA); rabbit MDC1 Sigma cat# PLA0016 (WB, IF, and PLA); rabbit MCM4 Cell Signaling Technology cat# 12973 (WB); mouse Actin-HRP Sigma cat# A3854 (WB); [ ] mouse Lamin A cat# SAB4200420 (WB); mouse PARP1 BD Biosciences cat# 51-6639GR (WB); goat 53BP1 Sigma cat# PLA0303 (PLA and IP); goat anti-mouse HRP Sigma cat# A3682 (WB); goat anti-rabbit Proteintech cat# SA00001-2 (WB); mouse Cyclin A Santa Cruz Biotechnology cat# sc-271682 (WB); rabbit Cyclin A Cell Signaling Technology cat# 67955S (IF); mouse Cyclin B Santa Cruz Biotechnology cat# sc-245 (WB); rabbit p21 Cell Signaling Technology cat# 2947S (WB); γH2AX phospho-Ser139 Cell Signaling Technology cat# 9718S (WB and IF); rabbit Poly ADP-Ribose Cell Signaling Technology cat# 83732S (WB); mouse MDM2 SMP14 Santa Cruz Biotechnology cat# sc-965 (IP); mouse IgG Santa Cruz Biotechnology cat# sc-2025 (IP); Purified mouse MDM2 4B2 [ ]; and purified mouse MDM2 2A9 [ ] were used for PLA and IP and prepared as described [ ].

Techniques: Modification, shRNA, Expressing, Plasmid Preparation, SDS Page, Western Blot

Journal: Journal of Cell Science

Article Title: Ezrin directly interacts with AQP2 and promotes its endocytosis

doi: 10.1242/jcs.204842

Figure Lengend Snippet: VP treatment enhances apical AQP2 expression and its colocalization with ezrin. (A) VP treatment enhances apical AQP2 expression and its colocalization with ezrin in cultured renal epithelial cells. AQP2-MDCK cells were stained with antibodies against ezrin (green) and AQP2 (red) in the presence (VP) and absence (Control) of VP treatment (AVP 20 nM for 20 min). The larger panels represent confocal sections through the subapical regions of the cells above the nucleus. The smaller horizontal strips at the bottom of each panel are z-sections through the entire cell for direct comparison of the respective staining intensities of the apical and basolateral membranes, and the cytosol. Upper panels show that in the absence of VP stimulation, ezrin staining localized to the cytosol and basolateral region, while AQP2 staining was mainly detected in the subapical region. Lower panels show that after VP treatment, the ezrin signal was redistributed toward the apical and sub-apical regions and partially colocalized with the similarly apically redistributed AQP2. Scale bar: 10 μm. (B) Super-resolution Airyscan confocal microscopy imaging revealed that AQP2 and ezrin partially colocalize on the apical membrane in VP-treated MDCK cells. Left panels show no apparent colocalization of ezrin and AQP2, in the absence of VP stimulation. Right panels are cells treated with VP. Scale bar: 5 μm. (C) AQP2 and ezrin are co-expressed in principal cells of the Brattleboro rat collecting duct, and co-accumulate on the plasma membrane after vasopressin treatment. Without VP treatment (Control), ezrin was located in the cytosol and basal region, while AQP2 was detected mainly in the sub-apical region of the principal cells of the collecting ducts. After 7 days of VP treatment (VP), ezrin (red in the merge panel) colocalized with AQP2 (green in the merge panel) on the plasma membrane of the principal cells. Scale bar: 20 μm.

Article Snippet: The commercial primary antibodies are from the following vendors: rabbit polyclonal anti-AQP2 antibody (#AQP2-002, Alomone Labs, Jerusalem, Israel), goat polyclonal anti-AQP2 antibody (sc9882, Santa Cruz Biotechnology, Dallas, TX), rabbit polyclonal anti-ezrin (3145s, Cell Signaling), rabbit anti-AQP2 p256 (PhosphoSolutions, Aurora, CO, USA), rabbit anti-AQP2 p261 (PhosphoSolutions), mouse monoclonal anti-GST antibody (sc138, Santa Cruz Biotechnology), mouse monoclonal anti-His antibody (70796, EMD Millipore, Billerica, MA), mouse anti-GAPDH antibody (AM4300, Ambion/Thermo Fisher Scientific), mouse anti β-actin antibody (A5441, Sigma-Aldrich), and rabbit anti-transferrin receptor 1 antibody (ab84036, Abcam, Cambridge, MA).

Techniques: Expressing, Cell Culture, Staining, Confocal Microscopy, Imaging

Journal: Journal of Cell Science

Article Title: Ezrin directly interacts with AQP2 and promotes its endocytosis

doi: 10.1242/jcs.204842

Figure Lengend Snippet: Interaction of AQP2 with ezrin is detected in co-IP experiments. (A) List of ezrin peptides detected by mass spectrometry from the AQP2 co-IP complex. (B,C) By using an anti-ezrin antibody for co-IP, we were able to detect AQP2 in the co-IP complex from stable AQP2-expressing LLC-PK1 cell lysates and mouse kidney (B). Similarly, ezrin signal was detected in the co-IP complex using anti-AQP2 antibody (C). WB, western blotting.

Article Snippet: The commercial primary antibodies are from the following vendors: rabbit polyclonal anti-AQP2 antibody (#AQP2-002, Alomone Labs, Jerusalem, Israel), goat polyclonal anti-AQP2 antibody (sc9882, Santa Cruz Biotechnology, Dallas, TX), rabbit polyclonal anti-ezrin (3145s, Cell Signaling), rabbit anti-AQP2 p256 (PhosphoSolutions, Aurora, CO, USA), rabbit anti-AQP2 p261 (PhosphoSolutions), mouse monoclonal anti-GST antibody (sc138, Santa Cruz Biotechnology), mouse monoclonal anti-His antibody (70796, EMD Millipore, Billerica, MA), mouse anti-GAPDH antibody (AM4300, Ambion/Thermo Fisher Scientific), mouse anti β-actin antibody (A5441, Sigma-Aldrich), and rabbit anti-transferrin receptor 1 antibody (ab84036, Abcam, Cambridge, MA).

Techniques: Co-Immunoprecipitation Assay, Mass Spectrometry, Expressing, Western Blot

Journal: Journal of Cell Science

Article Title: Ezrin directly interacts with AQP2 and promotes its endocytosis

doi: 10.1242/jcs.204842

Figure Lengend Snippet: AQP2 interacts with ezrin via the ezrin N-terminal FERM-containing domain. (A) Recombinant His-tagged ezrin full-length (FL, amino acids 1–586), N-terminus (NT, amino acids 1–308), and C-terminus (CT, amino acids 285–586) were expressed in E. coli and purified to homogeneity as revealed by SDS-PAGE together with purified recombinant AQP2 C-terminus (AQP2CT). Schematic representation of each recombinant protein is shown in B. (C) The purified His-tagged ezrin full-length protein (FL) and the N-terminal FERM-containing recombinant protein (but not the C-terminal protein) were able to pull down AQP2 from both LLC-AQP2 cell lysates and mouse kidney lysates. Lanes 1–3, beads alone pulled down with PBS (lane 1), LLC-AQP2 cell lysate (lane 2) and kidney lysate (lane 3); lanes 4–6, ezrin FL pulled down with PBS (lane 4), LLC-AQP2 cell lysate (lane 5) and kidney lysate (lane 6); lanes 7–9, ezrin NT pulled down with PBS (lane 7), LLC-AQP2 cell lysate (lane 8) and kidney lysate (lane 9); lanes 10–12, ezrin CT pulled down with PBS (lane 10), LLC-AQP2 cell lysate (lane 11) and kidney lysate (lane 12). WB, western blotting.

Article Snippet: The commercial primary antibodies are from the following vendors: rabbit polyclonal anti-AQP2 antibody (#AQP2-002, Alomone Labs, Jerusalem, Israel), goat polyclonal anti-AQP2 antibody (sc9882, Santa Cruz Biotechnology, Dallas, TX), rabbit polyclonal anti-ezrin (3145s, Cell Signaling), rabbit anti-AQP2 p256 (PhosphoSolutions, Aurora, CO, USA), rabbit anti-AQP2 p261 (PhosphoSolutions), mouse monoclonal anti-GST antibody (sc138, Santa Cruz Biotechnology), mouse monoclonal anti-His antibody (70796, EMD Millipore, Billerica, MA), mouse anti-GAPDH antibody (AM4300, Ambion/Thermo Fisher Scientific), mouse anti β-actin antibody (A5441, Sigma-Aldrich), and rabbit anti-transferrin receptor 1 antibody (ab84036, Abcam, Cambridge, MA).

Techniques: Recombinant, Purification, SDS Page, Western Blot

Journal: Journal of Cell Science

Article Title: Ezrin directly interacts with AQP2 and promotes its endocytosis

doi: 10.1242/jcs.204842

Figure Lengend Snippet: AQP2 C-terminus directly interacts with ezrin N-terminal FERM-containing domain. The direct interaction of AQP2 and ezrin is revealed by pulldown experiments using purified recombinant ezrin and AQP2 proteins. Only the purified ezrin full-length (FL) and the N-terminus FERM domain-containing (NT) recombinant protein were able to pull down the purified AQP2 C-terminus. The ezrin C-terminal domain (CT) did not pull down the AQP2 C-terminal domain. WB, western blotting.

Article Snippet: The commercial primary antibodies are from the following vendors: rabbit polyclonal anti-AQP2 antibody (#AQP2-002, Alomone Labs, Jerusalem, Israel), goat polyclonal anti-AQP2 antibody (sc9882, Santa Cruz Biotechnology, Dallas, TX), rabbit polyclonal anti-ezrin (3145s, Cell Signaling), rabbit anti-AQP2 p256 (PhosphoSolutions, Aurora, CO, USA), rabbit anti-AQP2 p261 (PhosphoSolutions), mouse monoclonal anti-GST antibody (sc138, Santa Cruz Biotechnology), mouse monoclonal anti-His antibody (70796, EMD Millipore, Billerica, MA), mouse anti-GAPDH antibody (AM4300, Ambion/Thermo Fisher Scientific), mouse anti β-actin antibody (A5441, Sigma-Aldrich), and rabbit anti-transferrin receptor 1 antibody (ab84036, Abcam, Cambridge, MA).

Techniques: Purification, Recombinant, Western Blot

Journal: Journal of Cell Science

Article Title: Ezrin directly interacts with AQP2 and promotes its endocytosis

doi: 10.1242/jcs.204842

Figure Lengend Snippet: Downregulating ezrin causes membrane accumulation of AQP2. (A) Immunoblotting revealed a strong knockdown of endogenous ezrin in LLC-AQP2 cells by ezrin shRNA lentivirus. There was an ∼80% reduction of endogenous ezrin in ezrin shRNA knockdown LLC-AQP2 cells (graph shows the quantification of ezrin band intensity relative to actin). Results are mean±s.e.m. (n≥3). ***P<0.001 (one-way ANOVA). (B) Immunofluorescence staining of AQP2 in control LLC-AQP2 cells and cells infected with ezrin shRNA lentivirus. After knocking down ezrin in LLC-AQP2 cells, AQP2 was found to increasingly accumulate on the cell surface under baseline conditions (without any stimulation). VP-treated LLC-AQP2 cells were used for comparison. Scale bar: 10 μm. (C) Surface biotinylation experiment revealed a significantly increased accumulation of AQP2 signal on cell surface after knocking down ezrin expression in cells. Transferrin receptor 1 (TFR-1) was used as an internal control for stable membrane proteins. WB, western blotting.

Article Snippet: The commercial primary antibodies are from the following vendors: rabbit polyclonal anti-AQP2 antibody (#AQP2-002, Alomone Labs, Jerusalem, Israel), goat polyclonal anti-AQP2 antibody (sc9882, Santa Cruz Biotechnology, Dallas, TX), rabbit polyclonal anti-ezrin (3145s, Cell Signaling), rabbit anti-AQP2 p256 (PhosphoSolutions, Aurora, CO, USA), rabbit anti-AQP2 p261 (PhosphoSolutions), mouse monoclonal anti-GST antibody (sc138, Santa Cruz Biotechnology), mouse monoclonal anti-His antibody (70796, EMD Millipore, Billerica, MA), mouse anti-GAPDH antibody (AM4300, Ambion/Thermo Fisher Scientific), mouse anti β-actin antibody (A5441, Sigma-Aldrich), and rabbit anti-transferrin receptor 1 antibody (ab84036, Abcam, Cambridge, MA).

Techniques: Western Blot, shRNA, Immunofluorescence, Staining, Infection, Expressing

Journal: Journal of Cell Science

Article Title: Ezrin directly interacts with AQP2 and promotes its endocytosis

doi: 10.1242/jcs.204842

Figure Lengend Snippet: Phosphorylation of key residues in AQP2 was not altered in ezrin knockdown cells. (A) Representative western blot showing that there is no alteration of the levels of total AQP2 and AQP2 phosphorylated at residues 256 or 261 in ezrin shRNA lentivirus-infected LLC-AQP2 cells unlike in cells treated with VP. Immunoblot using anti-β-actin antibody was used as control. Quantification of western blotting results for AQP2 phosphorylated on S256 (p256), S261 (p261) and total AQP2. (B) Intracellular cAMP measurement in LLC-PK1 cells. The intracellular cAMP concentration was significantly increased by 30 min VP treatment (20 nM LVP) without clonal variation. After ezrin knockdown no significant difference in cAMP concentration was observed compared to control. Results in A and B are mean±s.e.m. (n≥3). **P<0.01, ***P<0.001 (one-way ANOVA).

Article Snippet: The commercial primary antibodies are from the following vendors: rabbit polyclonal anti-AQP2 antibody (#AQP2-002, Alomone Labs, Jerusalem, Israel), goat polyclonal anti-AQP2 antibody (sc9882, Santa Cruz Biotechnology, Dallas, TX), rabbit polyclonal anti-ezrin (3145s, Cell Signaling), rabbit anti-AQP2 p256 (PhosphoSolutions, Aurora, CO, USA), rabbit anti-AQP2 p261 (PhosphoSolutions), mouse monoclonal anti-GST antibody (sc138, Santa Cruz Biotechnology), mouse monoclonal anti-His antibody (70796, EMD Millipore, Billerica, MA), mouse anti-GAPDH antibody (AM4300, Ambion/Thermo Fisher Scientific), mouse anti β-actin antibody (A5441, Sigma-Aldrich), and rabbit anti-transferrin receptor 1 antibody (ab84036, Abcam, Cambridge, MA).

Techniques: Western Blot, shRNA, Infection, Concentration Assay

Journal: Journal of Cell Science

Article Title: Ezrin directly interacts with AQP2 and promotes its endocytosis

doi: 10.1242/jcs.204842

Figure Lengend Snippet: Downregulating ezrin reduces clathrin-mediated endocytosis and causes concomitant membrane accumulation of AQP2 without affecting the overall exocytosis. (A) Ezrin knockdown does not affect the overall exocytosis in LLC-AQP2-ssYFP cells. LLC-AQP2 cells were stably transfected with ssYFP. The fluorescence signal in the extracellular medium was measured in LLC-AQP2-ssYFP cells with and without treatment with VP or ezrin shRNA lentivirus, respectively. The fluorescence intensity in the medium reflected the exocytotic activity of LLC-AQP2-ssYFP cells, and therefore the rate of exocytosis. No significant increase was seen in the overall exocytosis in LLC-AQP2-ssYFP cells treated with ezrin shRNA lentivirus compared to control. In contrast, a significant increase in exocytosis was observed in VP-treated LLC-AQP2-ssYFP cells, which is consistent with our previous reports (Nunes et al., 2008). (B) Endocytosis assay using Rhodamine-conjugated transferrin showed that Alexa Fluor 568-labeled transferrin accumulated on the apical membrane following ezrin knockdown (upper panel), and a simultaneous acute membrane accumulation of AQP2 (lower panel). Scale bar: 10 μm. (C) A bar graph showing that ezrin knockdown affects clathrin-mediated endocytosis. A block of endocytosis with MβCD was used as a positive control. Results in A and C are mean±s.e.m. (n≥3). **P<0.01, ***P<0.001 (one-way ANOVA).

Article Snippet: The commercial primary antibodies are from the following vendors: rabbit polyclonal anti-AQP2 antibody (#AQP2-002, Alomone Labs, Jerusalem, Israel), goat polyclonal anti-AQP2 antibody (sc9882, Santa Cruz Biotechnology, Dallas, TX), rabbit polyclonal anti-ezrin (3145s, Cell Signaling), rabbit anti-AQP2 p256 (PhosphoSolutions, Aurora, CO, USA), rabbit anti-AQP2 p261 (PhosphoSolutions), mouse monoclonal anti-GST antibody (sc138, Santa Cruz Biotechnology), mouse monoclonal anti-His antibody (70796, EMD Millipore, Billerica, MA), mouse anti-GAPDH antibody (AM4300, Ambion/Thermo Fisher Scientific), mouse anti β-actin antibody (A5441, Sigma-Aldrich), and rabbit anti-transferrin receptor 1 antibody (ab84036, Abcam, Cambridge, MA).

Techniques: Stable Transfection, Transfection, Fluorescence, shRNA, Activity Assay, Endocytosis Assay, Labeling, Blocking Assay, Positive Control

Journal: Journal of Cell Science

Article Title: Ezrin directly interacts with AQP2 and promotes its endocytosis

doi: 10.1242/jcs.204842

Figure Lengend Snippet: AQP2 internalization was reduced after ezrin knockdown in a cold block experiment. (A) Dynamic distribution of AQP2 after a 20°C cold block was revealed by immunofluorescence staining of LLC-AQP2 cells. Cells were incubated at 20°C for 30, 60 and 120 min to block AQP2 release from the trans-Golgi network. AQP2 formed a ‘perinuclear patch’ in cells at 20°C. In the absence of protein synthesis (blocked by cycloheximide), the speed of formation and immunostaining intensity of the perinuclear patch reflect the speed and extent of AQP2 internalization from the cell surface over time. Scale bar: 10 μm. (B) Quantification of the growth of the AQP2-positive perinuclear patch after 20°C cold block over time. The fluorescence intensity of AQP2 staining in the perinuclear patch was measured using Volocity software as described previously (Arthur et al., 2015). Results are mean±s.e.m. (n≥13 for each data point). Experiments were repeated at least three times.

Article Snippet: The commercial primary antibodies are from the following vendors: rabbit polyclonal anti-AQP2 antibody (#AQP2-002, Alomone Labs, Jerusalem, Israel), goat polyclonal anti-AQP2 antibody (sc9882, Santa Cruz Biotechnology, Dallas, TX), rabbit polyclonal anti-ezrin (3145s, Cell Signaling), rabbit anti-AQP2 p256 (PhosphoSolutions, Aurora, CO, USA), rabbit anti-AQP2 p261 (PhosphoSolutions), mouse monoclonal anti-GST antibody (sc138, Santa Cruz Biotechnology), mouse monoclonal anti-His antibody (70796, EMD Millipore, Billerica, MA), mouse anti-GAPDH antibody (AM4300, Ambion/Thermo Fisher Scientific), mouse anti β-actin antibody (A5441, Sigma-Aldrich), and rabbit anti-transferrin receptor 1 antibody (ab84036, Abcam, Cambridge, MA).

Techniques: Blocking Assay, Immunofluorescence, Staining, Incubation, Immunostaining, Fluorescence, Software

Journal: Nature Communications

Article Title: BCAT1 controls metabolic reprogramming in activated human macrophages and is associated with inflammatory diseases

doi: 10.1038/ncomms16040

Figure Lengend Snippet: ( a ) RNA-sequencing in human monocyte-derived macrophages (hMDMs) shows mRNA copies of BCAT enzymes in normalized counts. n =14 healthy donor hMDMs were used for RNA-seq. ( b ) Chemical structure of ERG240 and leucine. ( c ) Enzymatic activity of recombinant human BCAT1 and BCAT2 in presence of ERG240. ( d ) Relative expression of IRG1 , HIF1A and IL1B measured by qRT-PCR in control, ERG240-treated (20 mM, 3 h), LPS treated (100 ng ml −1 , 3 h), and LPS+ERG240 treated (LPS, 100 ng ml −1 ; ERG240, 20 mM for 3 h) hMDMs. ns, non-significant. All expression values are normalized to those obtained for HPRT gene expression. n =3 healthy donor hMDMs were used in each group. ( e ) Western blot analysis of IRG1, IL-1β, HIF-1α and beta-actin (ACTB) in control (untreated), ERG240-treated (20 mM, 3 h), LPS treated (100 ng ml −1 , 3 h) and LPS+ERG240 treated (LPS, 100 ng ml −1 ; ERG240, 20 mM for 3 h) hMDMs. The experiment is representative of three independent experiments using n =3 healthy donor hMDMs each. ( f ) GC/MS results showing itaconic acid production in hMDMs in presence or absence of ERG240 (20 mM). LPS treatment was for 8 h (100 ng ml −1 ). GC/MS plot for each donor is shown separately where itaconate abundance denotes arbitrary units. Error bars are s.e.m. Significance was tested using two-tailed Student’s t -test or one-way ANOVA. * P <10 −3 following two-way ANOVA.

Article Snippet: Lipopolysaccharide was purchased from Sigma (LPS; Escherichia coli serotype 0111:B4; Cat No L4391) and the antibodies used in Western Blot analyses are as follows: IL-1β (Cell Signaling Technologies, #12703; D3U3E, dilution 1/1,000), IRG1 (Abcam, ab122624, rabbit polyclonal, dilution, 1/1,000), HIF-1α (NOVUS biological, NB100-449, rabbit polyclonal, dilution 1/2,000), ACTB (Santa Cruz, sc-47778, β-Actin (C4), mouse monoclonal, dilution 1/2,000), BCAT1 (anti Bcat1 raised in rabbit, produced in Susan Hutson’s laboratory , 8382, dilution 1/120,000), TUBB (Cell Signaling Technologies, #2128, rabbit monoclonal, dilution 1/30,000).

Techniques: RNA Sequencing, Derivative Assay, Activity Assay, Recombinant, Expressing, Quantitative RT-PCR, Control, Gene Expression, Western Blot, Gas Chromatography-Mass Spectrometry, Two Tailed Test

Journal: eLife

Article Title: Inhibition of DNMT1 methyltransferase activity via glucose-regulated O-GlcNAcylation alters the epigenome

doi: 10.7554/elife.85595

Figure Lengend Snippet: Figure 1. High glucose increases O-GlcNAcylation of DNMT1 in cell lines and primary cells. (A) Hep3B cells were treated with glucose (5 mM or 25 mM) with or without Thiamet-G (TMG). Shown are immunoblots of collected lysates using antibody targeting O-GlcNAc and GAPDH (n = 3). (B) Lysates of Hep3B treated with glucose were immunoprecipitated with DNMT1 and immunoprecipitates were immunoblotted with antibody targeting O-GlcNAc (n = 3). (C) Peripheral blood mononuclear cells (PBMCs) were isolated from three individual donor blood samples and treated with increasing concentration of glucose for 24 hr. Collected cell lysates from PBMCs were immunoprecipitated with antibody targeting DNMT1 and immunoblotted for O-GlcNAc. Representative blot from one donor (n = 3). (D) Immunoblots for O-GlcNAc and GAPDH from liver samples of C57BL/6J mice given a high- fat/high-sucrose diet (HF/HS) or normal diet (chow) for 4 mo, and immunoprecipitated with Dnmt1. Lysates of mouse liver were immunoprecipitated with Dnmt1 and immunoprecipitates were immunoblotted with antibody targeting O-GlcNAc. *p<0.001; **p<0.0005; ***p<0.0001 by Student’s t-test (A-D); ns, not significant; data are represented as mean ± SD from three replicates of each sample.

Article Snippet: Reagent type (species) or resource Designation Source or reference Identifiers Additional information Gene (Homo sapiens) DNMT1 HUGO Gene Nomenclature Committee HGNC:2976 - Cell line (H. sapiens) Hep 3B2.1–7 ATCC HB- 8064 - Cell line (H. sapiens) Hep G2 ATCC HB- 8065 - Transfected construct (H. sapiens) pcDNA3/Myc- DNMT1 Addgene Plasmid #36939 Antibody Anti- beta- actin (D6A8) (rabbit monoclonal) Cell Signaling Technology Cat# 8457 WB (1:1000) Antibody Anti- alpha- tubulin (11H10) (rabbit monoclonal) Cell Signaling Technology Cat# 2125 WB (1:1000) Antibody Anti- DNMT1 (60B1220.1) (mouse monoclonal) Novus Biologicals Cat# NB100- 56519 IP (1:250) WB (1:1000) Antibody Anti- DNMT1 (H- 12) (mouse monoclonal) Santa Cruz Biotechnology Cat# sc- 271729 WB (1:1000) Antibody Anti- gamma H2A.X (rabbit polyclonal) Abcam Cat# ab11174 IF (1:1000) Antibody Anti- GAPDH (rabbit monoclonal) Abcam Cat# ab181602 WB (1:1000) Antibody Anti- H3 (rabbit polyclonal) Abcam Cat# ab1791 WB (1:1000) Antibody Anti- Myc [Myc.A7] (mouse monoclonal) Abcam Cat# ab18185 IP (1:250) WB (1:1000) Antibody Anti- O- GlcNAc (RL2) (mouse monoclonal) Abcam Cat# ab2739 WB (1:1000) Antibody Aoat anti- rabbit IgG H&L (HRP) (goat polyclonal) Abcam Cat# ab6721 WB (1:5000) Antibody Goat anti- mouse IgG H&L (HRP) (goat polyclonal) Abcam Cat# ab6789 WB (1:5000) Antibody Goat anti- mouse IgG (H+L), Alexa 488 (goat polyclonal) Invitrogen Cat# A32723 WB (1:1000) Sequence- based reagent DNMT1- T158A This paper PCR primers agccccaggatt CGA aggaaaagcacc Sequence- based reagent DNMT1- T158A This paper PCR primers ggtgcttttcct TCG aatcctggggct Sequence- based reagent DNMT1- T616A This paper PCR primers gacaggggaccc GCG aaagccaccacc Sequence- based reagent DNMT1- T616A This paper PCR primers ggtggtggcttt CGC gggtcccctgtc Sequence- based reagent DNMT1- S878A This paper PCR primers gcgagattcgag GAG cctccaaaaacc Sequence- based reagent DNMT1- S878A This paper PCR primers ggtttttggagg CTC ctcgaatctcgc Sequence- based reagent DNMT1- S878D This paper PCR primers gcgagattcgag GAC cctccaaaaacc Sequence- based reagent DNMT1- S878D This paper PCR primers ggtttttggagg GTC ctcgaatctcgc Sequence- based reagent DNMT1- T882A This paper PCR primers tcccctccaaaa GCC cagccaacagag Sequence- based reagent DNMT1- T882A This paper PCR primers ctctgttggctg GGC ttttggagggga Commercial assay or kit Q5 Site- Directed Mutagenesis kit NEB Cat# E0554S - Shin et al. eLife 2023;12:e85595.

Techniques: Western Blot, Immunoprecipitation, Isolation, Concentration Assay

Journal: eLife

Article Title: Inhibition of DNMT1 methyltransferase activity via glucose-regulated O-GlcNAcylation alters the epigenome

doi: 10.7554/elife.85595

Figure Lengend Snippet: Figure 2. Identification of O-GlcNAcylated sites within DNMT1 by LC-MS/MS. (A) Schematic drawing of the DNMT1 O-GlcNAc-modified region enriched from Hep3B cells based on mass spectrometry (MS) data and tandem MS (MS/MS) peaks. FTMS+ p NSI full MS (400.0000–1600.0000). DQDYARFESPPKTQPTEDNKF (S9 HexNAc) – S878. (B) Schematic diagram of identified novel O-GlcNAcylated and phosphorylated sites within DNMT1 as determined via LC-MS/MS. DMAP, DNA methyltransferase associated protein-binding domain; PCNA, proliferating cell nuclear antigen-binding domain; NLS, nuclear localization sequences; RFTS, replication foci targeting sequence domain; BAH, bromo-adjacent homology domain. (C) Sequence conservation of S878 in vertebrates. (D) Each immunoprecipitated Myc-DNMT1 wild type and substituted mutants was immunoblotted with an O- GlcNAc antibody (n = 3). **p<0.0005; ***p<0.0001 by Student’s t-test (D); N.D., not detected, ns, not significant; data are represented as mean ± SD from three replicates of each sample.

Article Snippet: Reagent type (species) or resource Designation Source or reference Identifiers Additional information Gene (Homo sapiens) DNMT1 HUGO Gene Nomenclature Committee HGNC:2976 - Cell line (H. sapiens) Hep 3B2.1–7 ATCC HB- 8064 - Cell line (H. sapiens) Hep G2 ATCC HB- 8065 - Transfected construct (H. sapiens) pcDNA3/Myc- DNMT1 Addgene Plasmid #36939 Antibody Anti- beta- actin (D6A8) (rabbit monoclonal) Cell Signaling Technology Cat# 8457 WB (1:1000) Antibody Anti- alpha- tubulin (11H10) (rabbit monoclonal) Cell Signaling Technology Cat# 2125 WB (1:1000) Antibody Anti- DNMT1 (60B1220.1) (mouse monoclonal) Novus Biologicals Cat# NB100- 56519 IP (1:250) WB (1:1000) Antibody Anti- DNMT1 (H- 12) (mouse monoclonal) Santa Cruz Biotechnology Cat# sc- 271729 WB (1:1000) Antibody Anti- gamma H2A.X (rabbit polyclonal) Abcam Cat# ab11174 IF (1:1000) Antibody Anti- GAPDH (rabbit monoclonal) Abcam Cat# ab181602 WB (1:1000) Antibody Anti- H3 (rabbit polyclonal) Abcam Cat# ab1791 WB (1:1000) Antibody Anti- Myc [Myc.A7] (mouse monoclonal) Abcam Cat# ab18185 IP (1:250) WB (1:1000) Antibody Anti- O- GlcNAc (RL2) (mouse monoclonal) Abcam Cat# ab2739 WB (1:1000) Antibody Aoat anti- rabbit IgG H&L (HRP) (goat polyclonal) Abcam Cat# ab6721 WB (1:5000) Antibody Goat anti- mouse IgG H&L (HRP) (goat polyclonal) Abcam Cat# ab6789 WB (1:5000) Antibody Goat anti- mouse IgG (H+L), Alexa 488 (goat polyclonal) Invitrogen Cat# A32723 WB (1:1000) Sequence- based reagent DNMT1- T158A This paper PCR primers agccccaggatt CGA aggaaaagcacc Sequence- based reagent DNMT1- T158A This paper PCR primers ggtgcttttcct TCG aatcctggggct Sequence- based reagent DNMT1- T616A This paper PCR primers gacaggggaccc GCG aaagccaccacc Sequence- based reagent DNMT1- T616A This paper PCR primers ggtggtggcttt CGC gggtcccctgtc Sequence- based reagent DNMT1- S878A This paper PCR primers gcgagattcgag GAG cctccaaaaacc Sequence- based reagent DNMT1- S878A This paper PCR primers ggtttttggagg CTC ctcgaatctcgc Sequence- based reagent DNMT1- S878D This paper PCR primers gcgagattcgag GAC cctccaaaaacc Sequence- based reagent DNMT1- S878D This paper PCR primers ggtttttggagg GTC ctcgaatctcgc Sequence- based reagent DNMT1- T882A This paper PCR primers tcccctccaaaa GCC cagccaacagag Sequence- based reagent DNMT1- T882A This paper PCR primers ctctgttggctg GGC ttttggagggga Commercial assay or kit Q5 Site- Directed Mutagenesis kit NEB Cat# E0554S - Shin et al. eLife 2023;12:e85595.

Techniques: Liquid Chromatography with Mass Spectroscopy, Modification, Mass Spectrometry, Tandem Mass Spectroscopy, Protein Binding, Binding Assay, Sequencing, Immunoprecipitation

Journal: eLife

Article Title: Inhibition of DNMT1 methyltransferase activity via glucose-regulated O-GlcNAcylation alters the epigenome

doi: 10.7554/elife.85595

Figure Lengend Snippet: Figure 4. High glucose leads to loss of DNA methylation at cancer-specific partially methylated domains (PMDs). (A) Density plot of DNA methylation for DNMT1-WT and DNMT1-S878A cells with either low (5 mM, CTRL) or high glucose/Thiamet-G (TMG) (25 mM, O-GlcNAc). (B) Genome browser screenshot of DNA methylation for DNMT1-WT and DNMT1-S878A cells and low or high glucose along with liver tumor PMDs from Li et al., 2016. (C) Boxplots of DNA methylation at PMDs or general genomic background (BG) for each DNMT1-WT and DNMT1-S878A treated with low (5 mM, CTRL) or high glucose/TMG (25 mM, O-GlcNAc). (D) Heatmap representation of global DNA methylation for DNMT1-WT and DNMT1-S878A cells under low (5 mM, CTRL) or high glucose/TMG (25 mM, O-GlcNAc) at gene-poor and gene-rich regions. (E) Methylation changes from O-GlcNAcylation of DNMT1 by wave score for replication timing (Hansen et al., 2010; Thurman et al., 2007). ***p<0.0001 by Wilcoxon signed-rank test (C).

Article Snippet: Reagent type (species) or resource Designation Source or reference Identifiers Additional information Gene (Homo sapiens) DNMT1 HUGO Gene Nomenclature Committee HGNC:2976 - Cell line (H. sapiens) Hep 3B2.1–7 ATCC HB- 8064 - Cell line (H. sapiens) Hep G2 ATCC HB- 8065 - Transfected construct (H. sapiens) pcDNA3/Myc- DNMT1 Addgene Plasmid #36939 Antibody Anti- beta- actin (D6A8) (rabbit monoclonal) Cell Signaling Technology Cat# 8457 WB (1:1000) Antibody Anti- alpha- tubulin (11H10) (rabbit monoclonal) Cell Signaling Technology Cat# 2125 WB (1:1000) Antibody Anti- DNMT1 (60B1220.1) (mouse monoclonal) Novus Biologicals Cat# NB100- 56519 IP (1:250) WB (1:1000) Antibody Anti- DNMT1 (H- 12) (mouse monoclonal) Santa Cruz Biotechnology Cat# sc- 271729 WB (1:1000) Antibody Anti- gamma H2A.X (rabbit polyclonal) Abcam Cat# ab11174 IF (1:1000) Antibody Anti- GAPDH (rabbit monoclonal) Abcam Cat# ab181602 WB (1:1000) Antibody Anti- H3 (rabbit polyclonal) Abcam Cat# ab1791 WB (1:1000) Antibody Anti- Myc [Myc.A7] (mouse monoclonal) Abcam Cat# ab18185 IP (1:250) WB (1:1000) Antibody Anti- O- GlcNAc (RL2) (mouse monoclonal) Abcam Cat# ab2739 WB (1:1000) Antibody Aoat anti- rabbit IgG H&L (HRP) (goat polyclonal) Abcam Cat# ab6721 WB (1:5000) Antibody Goat anti- mouse IgG H&L (HRP) (goat polyclonal) Abcam Cat# ab6789 WB (1:5000) Antibody Goat anti- mouse IgG (H+L), Alexa 488 (goat polyclonal) Invitrogen Cat# A32723 WB (1:1000) Sequence- based reagent DNMT1- T158A This paper PCR primers agccccaggatt CGA aggaaaagcacc Sequence- based reagent DNMT1- T158A This paper PCR primers ggtgcttttcct TCG aatcctggggct Sequence- based reagent DNMT1- T616A This paper PCR primers gacaggggaccc GCG aaagccaccacc Sequence- based reagent DNMT1- T616A This paper PCR primers ggtggtggcttt CGC gggtcccctgtc Sequence- based reagent DNMT1- S878A This paper PCR primers gcgagattcgag GAG cctccaaaaacc Sequence- based reagent DNMT1- S878A This paper PCR primers ggtttttggagg CTC ctcgaatctcgc Sequence- based reagent DNMT1- S878D This paper PCR primers gcgagattcgag GAC cctccaaaaacc Sequence- based reagent DNMT1- S878D This paper PCR primers ggtttttggagg GTC ctcgaatctcgc Sequence- based reagent DNMT1- T882A This paper PCR primers tcccctccaaaa GCC cagccaacagag Sequence- based reagent DNMT1- T882A This paper PCR primers ctctgttggctg GGC ttttggagggga Commercial assay or kit Q5 Site- Directed Mutagenesis kit NEB Cat# E0554S - Shin et al. eLife 2023;12:e85595.

Techniques: DNA Methylation Assay, Methylation

Journal: eLife

Article Title: Inhibition of DNMT1 methyltransferase activity via glucose-regulated O-GlcNAcylation alters the epigenome

doi: 10.7554/elife.85595

Figure Lengend Snippet: Figure 5. High glucose-induced reactive oxygen species (ROS) and DNA damage cause apoptotic cell death in DNMT1-WT cells. (A) Quantitative fluorescence image of ROS in DNMT1-WT and DNMT1-S878A cells with either low (5 mM, CTRL) or high glucose/Thiamet-G (TMG) (25 mM, O-GlcNAc). (B) Quantitative fluorescence image of γ-H2A.X in DNMT1-WT and DNMT1-S878A cells treated with low (5 mM, CTRL) or high glucose/TMG (25 mM, O- GlcNAc). (C) Quantitative fluorescence image of cell death in propidium iodide staining of DNMT1-WT and DNMT1-S878A cells under low (5 mM, CTRL) or high glucose/TMG (25 mM, O-GlcNAc). *p<0.001; **p<0.0005; ***p<0.0001 by Student’s t-test (A–C); data are represented as mean ± SD from three replicates of each sample.

Article Snippet: Reagent type (species) or resource Designation Source or reference Identifiers Additional information Gene (Homo sapiens) DNMT1 HUGO Gene Nomenclature Committee HGNC:2976 - Cell line (H. sapiens) Hep 3B2.1–7 ATCC HB- 8064 - Cell line (H. sapiens) Hep G2 ATCC HB- 8065 - Transfected construct (H. sapiens) pcDNA3/Myc- DNMT1 Addgene Plasmid #36939 Antibody Anti- beta- actin (D6A8) (rabbit monoclonal) Cell Signaling Technology Cat# 8457 WB (1:1000) Antibody Anti- alpha- tubulin (11H10) (rabbit monoclonal) Cell Signaling Technology Cat# 2125 WB (1:1000) Antibody Anti- DNMT1 (60B1220.1) (mouse monoclonal) Novus Biologicals Cat# NB100- 56519 IP (1:250) WB (1:1000) Antibody Anti- DNMT1 (H- 12) (mouse monoclonal) Santa Cruz Biotechnology Cat# sc- 271729 WB (1:1000) Antibody Anti- gamma H2A.X (rabbit polyclonal) Abcam Cat# ab11174 IF (1:1000) Antibody Anti- GAPDH (rabbit monoclonal) Abcam Cat# ab181602 WB (1:1000) Antibody Anti- H3 (rabbit polyclonal) Abcam Cat# ab1791 WB (1:1000) Antibody Anti- Myc [Myc.A7] (mouse monoclonal) Abcam Cat# ab18185 IP (1:250) WB (1:1000) Antibody Anti- O- GlcNAc (RL2) (mouse monoclonal) Abcam Cat# ab2739 WB (1:1000) Antibody Aoat anti- rabbit IgG H&L (HRP) (goat polyclonal) Abcam Cat# ab6721 WB (1:5000) Antibody Goat anti- mouse IgG H&L (HRP) (goat polyclonal) Abcam Cat# ab6789 WB (1:5000) Antibody Goat anti- mouse IgG (H+L), Alexa 488 (goat polyclonal) Invitrogen Cat# A32723 WB (1:1000) Sequence- based reagent DNMT1- T158A This paper PCR primers agccccaggatt CGA aggaaaagcacc Sequence- based reagent DNMT1- T158A This paper PCR primers ggtgcttttcct TCG aatcctggggct Sequence- based reagent DNMT1- T616A This paper PCR primers gacaggggaccc GCG aaagccaccacc Sequence- based reagent DNMT1- T616A This paper PCR primers ggtggtggcttt CGC gggtcccctgtc Sequence- based reagent DNMT1- S878A This paper PCR primers gcgagattcgag GAG cctccaaaaacc Sequence- based reagent DNMT1- S878A This paper PCR primers ggtttttggagg CTC ctcgaatctcgc Sequence- based reagent DNMT1- S878D This paper PCR primers gcgagattcgag GAC cctccaaaaacc Sequence- based reagent DNMT1- S878D This paper PCR primers ggtttttggagg GTC ctcgaatctcgc Sequence- based reagent DNMT1- T882A This paper PCR primers tcccctccaaaa GCC cagccaacagag Sequence- based reagent DNMT1- T882A This paper PCR primers ctctgttggctg GGC ttttggagggga Commercial assay or kit Q5 Site- Directed Mutagenesis kit NEB Cat# E0554S - Shin et al. eLife 2023;12:e85595.

Techniques: Fluorescence, Staining

Journal:

Article Title: The Pharmacological Chaperone 1-Deoxygalactonojirimycin Reduces Tissue Globotriaosylceramide Levels in a Mouse Model of Fabry Disease

doi: 10.1038/mt.2009.220

Figure Lengend Snippet: hR301Q α-Gal A Tg/KO mice have reduced α-Gal A and elevated GL-3 levels. (a) α-Gal A and GL-3 levels were assessed in tissue lysates prepared from skin, heart, and kidney of 12-week-old male GLA KO (open bars), hR301Q α-Gal A Tg/KO (gray bars), and wild-type C57BL/6 (black bars) mice. α-Gal A activity in hR301Q α-Gal A Tg/KO and GLA KO mice was significantly lower than that of wild-type mice (*P < 0.05, t-test); α-Gal A activity in hR301Q α-Gal A Tg/KO mice was significantly higher than that of GLA KO mice (#P < 0.05, t-test). GL-3 levels in hR301Q α-Gal A Tg/KO and GLA KO mice were significantly higher than those of wild-type mice (*P < 0.05, t-test). Each bar represents the mean ± SEM from four mice per group analyzed in triplicate. (b) Cell-type specific GL-3 staining was assessed by immunohistochemistry in GLA KO, hR301Q α-Gal A Tg/KO, and wild-type C57BL/6 mouse tissues. GL-3 staining is represented as brown spots (black arrows); nuclei are represented as green spots (stained with methyl green). The data shown are representative photomicrographs from four 12-week-old male mice using ×20 magnification, except for the glomeruli, which are representative of four 7-month-old male mice using ×40 magnification. α-Gal A, α-galactosidase A; GL-3, globotriaosylceramide; KO, knockout; Tg, transgenic.

Article Snippet: 49 Western blotting of tissue α-Gal A. Tissue lysates (50 µg total protein) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis on 12% polyacrylamide gels (Bio-Rad, Hercules, CA), transferred to polyvinylidene fluoride membranes (Bio-Rad), and immunoblotted with rabbit antihuman α-Gal A primary antibody (1:1,000 dilution).

Techniques: Activity Assay, Staining, Immunohistochemistry, Knock-Out, Transgenic Assay

Journal:

Article Title: The Pharmacological Chaperone 1-Deoxygalactonojirimycin Reduces Tissue Globotriaosylceramide Levels in a Mouse Model of Fabry Disease

doi: 10.1038/mt.2009.220

Figure Lengend Snippet: DGJ administration increases α-Gal A activity and reduces GL-3 in hR301Q α-Gal A Tg/KO mice. Eight-week-old male hR301Q α-Gal A Tg/KO mice were administered DGJ ad libitum in drinking water for 4 weeks at the indicated doses. Lysates from skin, heart, and kidney were subsequently tested for (a) α-Gal A activity and (b,c) GL-3 levels. (a) Significant increases in α-Gal A activity were seen in all three tissues (*P < 0.05 versus untreated, t-test). The effect was also significant for a linear trend, indicating a dose-dependent increase in α-Gal A activity (post hoc analysis, P < 0.05). Insets: Tissue α-Gal A protein levels were determined by western blotting. A dose-dependent increase in the 46 kd mature form of α-Gal A was seen in all tissues. Each lane on the blot contains tissue lysate from a single animal. The western blot shown is representative of two experiments. (b) Significant reductions in GL-3 levels were seen in all three tissues as measured by LC-MS/MS (*P < 0.05 versus untreated, t-test). Blue bars (a,b) represent α-Gal A and GL-3 levels from age-matched untreated wild-type C57BL/6 mice. Each bar represents pooled data from three independent studies with the mean ± SEM of 21 hR301Q α-Gal A Tg/KO mice/group or four untreated wild-type mice, all analyzed in triplicate. (c) Cell type–specific reduction of GL-3 in hR301Q α-Gal A Tg/KO mice administered DGJ (100 mg/kg per day) ad libitum in drinking water was assessed by immunohistochemistry. GL-3 staining is represented as brown spots denoted with black arrows. The data shown are representative photomicrographs from 7–8 mice/group (magnification: ×20, insets: ×40). α-Gal A, α-galactosidase A; DGJ, 1-deoxygalactonojirimycin; GL-3, globotriaosylceramide; KO, knockout; Tg, transgenic.

Article Snippet: 49 Western blotting of tissue α-Gal A. Tissue lysates (50 µg total protein) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis on 12% polyacrylamide gels (Bio-Rad, Hercules, CA), transferred to polyvinylidene fluoride membranes (Bio-Rad), and immunoblotted with rabbit antihuman α-Gal A primary antibody (1:1,000 dilution).

Techniques: Activity Assay, Western Blot, Liquid Chromatography with Mass Spectroscopy, Immunohistochemistry, Staining, Knock-Out, Transgenic Assay

Journal:

Article Title: The Pharmacological Chaperone 1-Deoxygalactonojirimycin Reduces Tissue Globotriaosylceramide Levels in a Mouse Model of Fabry Disease

doi: 10.1038/mt.2009.220

Figure Lengend Snippet: Long-term (24-week) administration of DGJ results in greater GL-3 reduction compared to 4-week administration. (a) Four-week-old male hR301Q α-Gal A Tg/KO mice were administered DGJ ad libitum in drinking water for 24 weeks at the indicated doses. GL-3 levels were subsequently measured in skin, heart, and kidney lysates by LC-MS/MS. Significant reductions in GL-3 were seen in all three tissues (*P < 0.05 versus untreated, t-test). The effect was also significant for a linear trend, indicating a dose-dependent decrease in GL-3 levels (post hoc analysis, P < 0.05). Each bar represents the mean ± SEM of 7–8 mice/dose group analyzed in triplicate. (b) Cell type–specific GL-3 reductions were assessed by immunohistochemistry. GL-3 staining is represented as brown spots. Data shown are representative photomicrographs from 7–8 mice/group (magnification: ×20, except glomeruli: ×40). DGJ, 1-deoxygalactonojirimycin; GL-3, globotriaosylceramide; KO, knockout; Tg, transgenic.

Article Snippet: 49 Western blotting of tissue α-Gal A. Tissue lysates (50 µg total protein) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis on 12% polyacrylamide gels (Bio-Rad, Hercules, CA), transferred to polyvinylidene fluoride membranes (Bio-Rad), and immunoblotted with rabbit antihuman α-Gal A primary antibody (1:1,000 dilution).

Techniques: Liquid Chromatography with Mass Spectroscopy, Immunohistochemistry, Staining, Knock-Out, Transgenic Assay

Journal:

Article Title: The Pharmacological Chaperone 1-Deoxygalactonojirimycin Reduces Tissue Globotriaosylceramide Levels in a Mouse Model of Fabry Disease

doi: 10.1038/mt.2009.220

Figure Lengend Snippet: Effect of 4- and 24-week DGJ administration on tissue GL-3 levels in hR301Q α-Gal A Tg/KO mice

Article Snippet: 49 Western blotting of tissue α-Gal A. Tissue lysates (50 µg total protein) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis on 12% polyacrylamide gels (Bio-Rad, Hercules, CA), transferred to polyvinylidene fluoride membranes (Bio-Rad), and immunoblotted with rabbit antihuman α-Gal A primary antibody (1:1,000 dilution).

Techniques:

Journal:

Article Title: The Pharmacological Chaperone 1-Deoxygalactonojirimycin Reduces Tissue Globotriaosylceramide Levels in a Mouse Model of Fabry Disease

doi: 10.1038/mt.2009.220

Figure Lengend Snippet: Less frequent DGJ administration results in greater GL-3 reduction. (a) Eight-week-old male hR301Q α-Gal A Tg/KO mice were administered drinking water (red dotted lines) or DGJ [100 mg/kg per day (red solid lines) ad libitum in drinking water] for 4 weeks, followed by a washout period (drinking water only) of up to 7 days. Groups of mice were then euthanized on days 0, 1, 3, 5, or 7 after DGJ withdrawal. Skin, heart, and kidney were collected at each time point, and α-Gal A activity and DGJ levels (blue lines) were measured in tissue lysates. Significantly increased α-Gal A activity was sustained in all three tissues for up to 7 days (*P < 0.05 versus untreated, t-test). For the α-Gal A activity, each data point represents the mean ± SEM of 7–8 mice/time point analyzed in triplicate; data were normalized to untreated levels. For tissue DGJ levels, each data point represents the mean ± SEM for five mice/time point. The limit of quantitation for DGJ in skin, heart, and kidney was 6, 4, and 10 ng/g, respectively. (b) Eight-week-old male hR301Q α-Gal A Tg/KO mice were administered DGJ (300 mg/kg per day) ad libitum in drinking water for 4 weeks either daily or less frequently using four cycles of a “4 on/3 off” regimen. GL-3 levels were subsequently measured in lysates from skin, heart, and kidney by LC-MS/MS. Significant reductions in GL-3 levels were seen in all tissues (*P < 0.05 versus untreated, t-test). GL-3 levels in skin, heart, and kidney also showed significantly greater reductions with less frequent compared to daily administration (#P < 0.05 daily versus “4 on/3 off”, t-test). Each bar represents pooled data from three independent studies with the mean ± SEM of 21 mice/group analyzed in triplicate. DGJ, 1-deoxygalactonojirimycin; GL-3, globotriaosylceramide; KO, knockout; Tg, transgenic.

Article Snippet: 49 Western blotting of tissue α-Gal A. Tissue lysates (50 µg total protein) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis on 12% polyacrylamide gels (Bio-Rad, Hercules, CA), transferred to polyvinylidene fluoride membranes (Bio-Rad), and immunoblotted with rabbit antihuman α-Gal A primary antibody (1:1,000 dilution).

Techniques: Activity Assay, Quantitation Assay, Liquid Chromatography with Mass Spectroscopy, Knock-Out, Transgenic Assay

Journal:

Article Title: The Pharmacological Chaperone 1-Deoxygalactonojirimycin Reduces Tissue Globotriaosylceramide Levels in a Mouse Model of Fabry Disease

doi: 10.1038/mt.2009.220

Figure Lengend Snippet: Long-term DGJ administration reduces accumulated tissue GL-3 in aged hR301Q α-Gal A Tg/KO mice. Twenty-four-week-old male hR301Q α-Gal A Tg/KO mice were administered DGJ ad libitum in drinking water for 24 weeks either daily or less frequently using a “4 on/3 off” regimen at the indicated doses. GL-3 levels were measured in lysates of skin, heart, and kidney by LC-MS/MS. Dose-dependent and significant reductions in GL-3 were seen in all three tissues (*P < 0.05 versus untreated, t-test). Reduction of GL-3 in skin and heart was significantly greater after less frequent compared to daily administration of 300 mg/kg DGJ (#P < 0.05 daily versus “4 on/3 off”, t-test). Each bar represents the mean ± SEM of 7–8 mice/group analyzed in triplicate. DGJ, 1-deoxygalactonojirimycin; GL-3, globotriaosylceramide; KO, knockout; Tg, transgenic.

Article Snippet: 49 Western blotting of tissue α-Gal A. Tissue lysates (50 µg total protein) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis on 12% polyacrylamide gels (Bio-Rad, Hercules, CA), transferred to polyvinylidene fluoride membranes (Bio-Rad), and immunoblotted with rabbit antihuman α-Gal A primary antibody (1:1,000 dilution).

Techniques: Liquid Chromatography with Mass Spectroscopy, Knock-Out, Transgenic Assay

Journal:

Article Title: The Pharmacological Chaperone 1-Deoxygalactonojirimycin Reduces Tissue Globotriaosylceramide Levels in a Mouse Model of Fabry Disease

doi: 10.1038/mt.2009.220

Figure Lengend Snippet: Effect of DGJ on plasma GL-3 levels in hR301Q α-Gal A Tg/KO mice

Article Snippet: 49 Western blotting of tissue α-Gal A. Tissue lysates (50 µg total protein) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis on 12% polyacrylamide gels (Bio-Rad, Hercules, CA), transferred to polyvinylidene fluoride membranes (Bio-Rad), and immunoblotted with rabbit antihuman α-Gal A primary antibody (1:1,000 dilution).

Techniques:

Journal:

Article Title: The Pharmacological Chaperone 1-Deoxygalactonojirimycin Reduces Tissue Globotriaosylceramide Levels in a Mouse Model of Fabry Disease

doi: 10.1038/mt.2009.220

Figure Lengend Snippet: DGJ increases α-Gal A and decreases GL-3 levels in brain of aged hR301Q α-Gal A Tg/KO mice. Twenty-four-week-old male hR301Q α-Gal A Tg/KO mice were administered DGJ ad libitum in drinking water for 12 weeks either daily or less frequently using a “4 on/3 off” regimen at the indicated doses. Brain α-Gal A and GL-3 levels were subsequently measured. Significant increases in α-Gal A and concomitant reductions in GL-3 were seen with DGJ administration (*P < 0.05 versus untreated, t-test). Each bar represents the mean ± SEM of 7–8 mice/group analyzed in triplicate. DGJ, 1-deoxygalactonojirimycin; GL-3, globotriaosylceramide; KO, knockout; Tg, transgenic.

Article Snippet: 49 Western blotting of tissue α-Gal A. Tissue lysates (50 µg total protein) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis on 12% polyacrylamide gels (Bio-Rad, Hercules, CA), transferred to polyvinylidene fluoride membranes (Bio-Rad), and immunoblotted with rabbit antihuman α-Gal A primary antibody (1:1,000 dilution).

Techniques: Knock-Out, Transgenic Assay

Journal: Cells

Article Title: Phosphorylation of PLK3 Is Controlled by Protein Phosphatase 6

doi: 10.3390/cells9061506

Figure Lengend Snippet: PLK3 localizes at plasma membrane and Golgi apparatus. ( A ) RPE cells were transfected with non-targeting control siRNA or two siRNAs targeting PLK3. After 48 h, whole cell lysates were separated by SDS-PAGE and probed with indicated antibodies. Numbers indicate mobility in kDa. Position of PLK3 is indicated by arrowheads. Importin was used as loading control. ( B ) RPE cells were transfected with non-targeting control siRNA or siRNA to PLK3 or PLK1. Whole cell lysates were probed with rabbit monoclonal PLK3 antibody (Cell Signaling). Protein 143−3− was used as loading control. The position of PLK3 is indicated by arrowheads. ( C ) HEK293 cells stably transfected with EGFP-PLK3 grown on coverslips were fixed and probed with antibodies against GM130 (Golgi marker) and γTubulin (centrosome marker) and imaged by confocal microscopy. The arrowhead indicates the position of the centrosome. A representative image is shown. Bar indicates 5 μm. ( D ) RPE cells transfected with EGFP-PLK3 were fixed and probed with antibodies against GM130 and γTubulin. The arrowhead indicates the position of the centrosome. A representative image is shown. Bar indicates 5 μm. ( E ) HEK293 cells expressing EGFP2−93 were fixed and probed with indicated antibodies. Bar indicates 5 μm. ( F ) Cell lysates from HEK293 and HEK293 transfected with EGFP-PLK3 or EGFP were probed with indicated antibodies by immunoblotting. Note that only PLK3 antibodies from Cell Signaling and St. Johns recognize overexpressed EGFP-PLK3. Antibody from St. Johns also shows a large number of cross-reacting bands. ( G ) HEK293 cells stably transfected with EGFP-PLK3 were fixed 2 h after exposure to mock or to UVC and imaged using confocal microscopy. Bar indicates 5 μm. ( H ) HEK293 cells stably transfected with EGFP-PLK3 were treated with leptomycin and proteasomal inhibitor MG132 for 3 h, fixed and imaged using confocal microscopy. ( I ) Cytosolic, nuclear soluble and chromatin fractions of RPE cells were probed with indicated antibodies. Tubulin is a marker of cytosol, CHK2 of the nuclear soluble fraction and histone H2AX of chromatin. Asterisk indicates non-specific band of PLK3 antibody.

Article Snippet: The following antibodies were used: rabbit monoclonal to PLK3 (clone D14F12, Cell Signaling Technology, #4896); rabbit polyclonal to PLK3 (Novus Biologicals, Abingdon, UK, NBP23-2530); goat polyclonal to PLK3 (Bio-Rad, Hercules, CA, USA, VPA00063); rabbit polyclonal to PLK3 (Sigma-Aldrich, St. Louis, MO, USA, HPA060318); mouse monoclonal to PLK3 (clone B37-2, BD Pharmingen, San Jose, CA, USA, 556518); rabbit polyclonal to PLK3 (St. Johns Laboratory, London, UK, STJ93099); rabbit monoclonal Phospho-PLK1 (Thr210) Antibody (Cell Signaling Technology, Danvers, MA, USA #9062); γ-tubulin (Sigma-Aldrich); GM130, p38-pT180/pY182, cJun-pSer73, phospho-histone H2AX-Ser139 (referred to as γ-H2AX) and HIF1α (Cell Signaling Technology); PPP6R1, PPP6R2 and PPP6R3 (A300-968A, A300-970A, A300-972A, Bethyl Laboratories, Montgomery, TX, USA); 14-3-3, mouse monoclonal to PPP6C and TFIIH (Santa Cruz Biotechnology, Dallas, TX, USA); and rabbit polyclonal to PPP6C (Abcam, Cambridge, UK).

Techniques: Clinical Proteomics, Membrane, Transfection, Control, SDS Page, Stable Transfection, Marker, Confocal Microscopy, Expressing, Western Blot

Journal: Cells

Article Title: Phosphorylation of PLK3 Is Controlled by Protein Phosphatase 6

doi: 10.3390/cells9061506

Figure Lengend Snippet: Distribution of EGFP-PLK3 during the cell cycle. ( A ) Cells were synchronized at G1/S transition with thymidine block, released to the fresh media containing nocodazole and collected in 2 h intervals. Last sample was collected by mitotic shake-off. Histone H3-pS10 is a marker of mitotic cells. Asterisk indicates non-specific reactivity. ( B ) HEK293 cells stably expressing EGFP-PLK3 were fixed, stained with an antibody against γ-tubulin, and analyzed by confocal microscopy. Maximal projections of representative cells in various phases of the cell cycle are shown. Bar indicates 10 μm.

Article Snippet: The following antibodies were used: rabbit monoclonal to PLK3 (clone D14F12, Cell Signaling Technology, #4896); rabbit polyclonal to PLK3 (Novus Biologicals, Abingdon, UK, NBP23-2530); goat polyclonal to PLK3 (Bio-Rad, Hercules, CA, USA, VPA00063); rabbit polyclonal to PLK3 (Sigma-Aldrich, St. Louis, MO, USA, HPA060318); mouse monoclonal to PLK3 (clone B37-2, BD Pharmingen, San Jose, CA, USA, 556518); rabbit polyclonal to PLK3 (St. Johns Laboratory, London, UK, STJ93099); rabbit monoclonal Phospho-PLK1 (Thr210) Antibody (Cell Signaling Technology, Danvers, MA, USA #9062); γ-tubulin (Sigma-Aldrich); GM130, p38-pT180/pY182, cJun-pSer73, phospho-histone H2AX-Ser139 (referred to as γ-H2AX) and HIF1α (Cell Signaling Technology); PPP6R1, PPP6R2 and PPP6R3 (A300-968A, A300-970A, A300-972A, Bethyl Laboratories, Montgomery, TX, USA); 14-3-3, mouse monoclonal to PPP6C and TFIIH (Santa Cruz Biotechnology, Dallas, TX, USA); and rabbit polyclonal to PPP6C (Abcam, Cambridge, UK).

Techniques: Blocking Assay, Marker, Stable Transfection, Expressing, Staining, Confocal Microscopy

Journal: Cells

Article Title: Phosphorylation of PLK3 Is Controlled by Protein Phosphatase 6

doi: 10.3390/cells9061506

Figure Lengend Snippet: Generation of RPE-PLK3 knock-out cells. ( A ) Schematic diagram of CRISPR/Cas9-mediated targeting of human PLK3 and of domain organization of PLK3 protein. Black arrowhead indicates the position of the critical ATP-binding Lysine 91 in the catalytic domain. Empty arrowhead indicates the position of the epitope of the PLK3 antibody from Cell Signaling. Fragments of the genomic DNA from selected clones were PCR amplified and analyzed by sequencing. ( B ) Parental RPE and selected clones of RPE-PLK3-KO cells were analyzed by immunoblotting using rabbit monoclonal PLK3 antibody (Cell Signalling). ( C ) Parental RPE and two independent clones of RP-PLK3-KO cells were probed with indicated antibodies. The position of PLK3 is indicated by an arrowhead. Please note the missing band corresponding to PLK3 in staining with antibody from St Johns laboratory and high level of cross-reactivity with other proteins.

Article Snippet: The following antibodies were used: rabbit monoclonal to PLK3 (clone D14F12, Cell Signaling Technology, #4896); rabbit polyclonal to PLK3 (Novus Biologicals, Abingdon, UK, NBP23-2530); goat polyclonal to PLK3 (Bio-Rad, Hercules, CA, USA, VPA00063); rabbit polyclonal to PLK3 (Sigma-Aldrich, St. Louis, MO, USA, HPA060318); mouse monoclonal to PLK3 (clone B37-2, BD Pharmingen, San Jose, CA, USA, 556518); rabbit polyclonal to PLK3 (St. Johns Laboratory, London, UK, STJ93099); rabbit monoclonal Phospho-PLK1 (Thr210) Antibody (Cell Signaling Technology, Danvers, MA, USA #9062); γ-tubulin (Sigma-Aldrich); GM130, p38-pT180/pY182, cJun-pSer73, phospho-histone H2AX-Ser139 (referred to as γ-H2AX) and HIF1α (Cell Signaling Technology); PPP6R1, PPP6R2 and PPP6R3 (A300-968A, A300-970A, A300-972A, Bethyl Laboratories, Montgomery, TX, USA); 14-3-3, mouse monoclonal to PPP6C and TFIIH (Santa Cruz Biotechnology, Dallas, TX, USA); and rabbit polyclonal to PPP6C (Abcam, Cambridge, UK).

Techniques: Knock-Out, CRISPR, Binding Assay, Clone Assay, Amplification, Sequencing, Western Blot, Staining

Journal: Cells

Article Title: Phosphorylation of PLK3 Is Controlled by Protein Phosphatase 6

doi: 10.3390/cells9061506

Figure Lengend Snippet: Loss of PLK3 does not affect cell response to various forms of stress. ( A ) RPE or RPE-PLK3-KO cells were exposed or not to UVC, collected after 30 min and whole cell lysates were analyzed by immunoblotting ( n = 3). Antibody against γH2AX was used as a marker of induced DNA damage and TFIIH was used as a loading control. ( B ) RPE or RPE-PLK3-KO cells were incubated with isotonic, hypertonic or hypotonic media for 20 min and whole cell lysates were analyzed by immunoblotting. Antibody against active the form of p38-pT180/pY182 was used as a marker of cellular stress and TFIIH as loading control ( n = 3). ( C ) RPE cells transfected with control or two various PLK3 siRNAs were treated as in A and analyzed by immunoblotting ( n = 2). ( D ) RPE cells transfected with control or two various PLK3 siRNAs were treated as in B and analyzed by immunoblotting ( n = 2). ( E ) Validation of PLK3 siRNA using RT-qPCR. PLK3 mRNA level was normalized to ATP5B. ( F ) RPE or RPE-PLK3-KO cells were treated or not with indicated doses of CoCl2 for 24 h and whole cell lysates were analyzed by immunoblotting ( n = 3). ( G ) RPE and HeLa cells transfected with control or two various PLK3 siRNAs were treated or not with CoCl2 for 24 h and analyzed by immunoblotting ( n = 2 for each cell type). ( H ) RPE or RPE-PLK3-KO cells were exposed or not to 3 Gy of ionizing radiation and fixed after 1 or 8 h. Number of γH2AX positive foci per nucleus was determined by ScanR microscopy. Lines indicate median. ( I ) RPE or RPE-PLK3-KO cells were treated as in H and whole cell lysates were analyzed by immunoblotting ( n = 2). ( J ) RPE cells transfected with control or two various PLK3 siRNAs were treated as in H and whole cell lysates were analyzed by immunoblotting ( n = 2). ( K ) Cells were transfected with the wild-type PLK3 or kinase-dead K91R mutant; PLK3 was isolated by GFP Trap and incubated with casein in kinase buffer supplemented with radioactive ATP. Phosphorylation of casein was detected by autoradiography. ( L ) Cells were exposed to various forms of stress including treatment with CoCl2 (300 μM, 12 h), exposure to UV (10 J/m 2 ), etoposide (4 μM, 1 h), hypertonic media (480 μM mannitol) or left untreated. EGFP-PLK3 was isolated using GFP Trap and kinase assay was performed as in H. Induction of stress pathways was analyzed by immunoblotting of cell extracts. Arrowhead indicates identical position on the electrophoretic gel. ( M ) Quantification of the kinase assay in L. Signal was normalized to the non-treated control. Plotted is median +/− SD ( n = 3). Statistical significance was evaluated by two-tail t -test and significance was set to p < 0.05. Ns. stands for non-significant.

Article Snippet: The following antibodies were used: rabbit monoclonal to PLK3 (clone D14F12, Cell Signaling Technology, #4896); rabbit polyclonal to PLK3 (Novus Biologicals, Abingdon, UK, NBP23-2530); goat polyclonal to PLK3 (Bio-Rad, Hercules, CA, USA, VPA00063); rabbit polyclonal to PLK3 (Sigma-Aldrich, St. Louis, MO, USA, HPA060318); mouse monoclonal to PLK3 (clone B37-2, BD Pharmingen, San Jose, CA, USA, 556518); rabbit polyclonal to PLK3 (St. Johns Laboratory, London, UK, STJ93099); rabbit monoclonal Phospho-PLK1 (Thr210) Antibody (Cell Signaling Technology, Danvers, MA, USA #9062); γ-tubulin (Sigma-Aldrich); GM130, p38-pT180/pY182, cJun-pSer73, phospho-histone H2AX-Ser139 (referred to as γ-H2AX) and HIF1α (Cell Signaling Technology); PPP6R1, PPP6R2 and PPP6R3 (A300-968A, A300-970A, A300-972A, Bethyl Laboratories, Montgomery, TX, USA); 14-3-3, mouse monoclonal to PPP6C and TFIIH (Santa Cruz Biotechnology, Dallas, TX, USA); and rabbit polyclonal to PPP6C (Abcam, Cambridge, UK).

Techniques: Western Blot, Marker, Control, Incubation, Transfection, Biomarker Discovery, Quantitative RT-PCR, Microscopy, Mutagenesis, Isolation, Phospho-proteomics, Autoradiography, Kinase Assay

Journal: Cells

Article Title: Phosphorylation of PLK3 Is Controlled by Protein Phosphatase 6

doi: 10.3390/cells9061506

Figure Lengend Snippet: PP6 phosphatase interacts with PLK3. ( A ) Lysates from HEK293 cells expressing EGFP or EGFP-PLK3 were incubated with GFP-Trap beads, washed three times and bound proteins were analyzed by mass spectrometry. Three biological replicates were performed. Numbers indicate average log difference between EGFP-PLK3 and EGFP. Listed are top 20 hits. ( B ) EGFP or EGFP-PLK3 was immunoprecipitated from stably transfected cells, and bound proteins were analysed by immunoblotting. Representative image is shown. ( C ) Lysates from HEK293 cells transfected with EGFP-PLK3, EGFP-PLK3-∆PBD or EGFP-PLK3-H590A-K592M (referred to as PLK3-HM) mutant were incubated with GFP-Trap and bound proteins were analysed by immunoblotting. ( D ) Cells expressing EGFP-PLK3 were transfected with control siRNA or siRNA to PP6C. After 48 h, cells were fixed, stained with antibodies against PP6C and γ-Tubulin and imaged using confocal microscopy. Arrowheads indicate the position of the centrosome. ( E ) Cells expressing EGFP-PLK3 were transfected with control siRNA or siRNA to PPP6R3. After 48 h, cells were fixed, stained with antibodies against PPP6R3 and γ-Tubulin, and imaged using confocal microscopy. Arrowheads indicate the position of the centrosome. ( F ) Validation of the knock-down efficiency in D and E. Cells transfected with control siRNA or siRNA to PPP6C, PPP6R1 or PPP6R3 were lysed and probed with indicated antibodies.

Article Snippet: The following antibodies were used: rabbit monoclonal to PLK3 (clone D14F12, Cell Signaling Technology, #4896); rabbit polyclonal to PLK3 (Novus Biologicals, Abingdon, UK, NBP23-2530); goat polyclonal to PLK3 (Bio-Rad, Hercules, CA, USA, VPA00063); rabbit polyclonal to PLK3 (Sigma-Aldrich, St. Louis, MO, USA, HPA060318); mouse monoclonal to PLK3 (clone B37-2, BD Pharmingen, San Jose, CA, USA, 556518); rabbit polyclonal to PLK3 (St. Johns Laboratory, London, UK, STJ93099); rabbit monoclonal Phospho-PLK1 (Thr210) Antibody (Cell Signaling Technology, Danvers, MA, USA #9062); γ-tubulin (Sigma-Aldrich); GM130, p38-pT180/pY182, cJun-pSer73, phospho-histone H2AX-Ser139 (referred to as γ-H2AX) and HIF1α (Cell Signaling Technology); PPP6R1, PPP6R2 and PPP6R3 (A300-968A, A300-970A, A300-972A, Bethyl Laboratories, Montgomery, TX, USA); 14-3-3, mouse monoclonal to PPP6C and TFIIH (Santa Cruz Biotechnology, Dallas, TX, USA); and rabbit polyclonal to PPP6C (Abcam, Cambridge, UK).

Techniques: Expressing, Incubation, Mass Spectrometry, Immunoprecipitation, Stable Transfection, Transfection, Western Blot, Mutagenesis, Control, Staining, Confocal Microscopy, Biomarker Discovery, Knockdown

Journal: Cells

Article Title: Phosphorylation of PLK3 Is Controlled by Protein Phosphatase 6

doi: 10.3390/cells9061506

Figure Lengend Snippet: PP6 controls the phosphorylation level of PLK3. ( A ) Conservation of the T-loop of human PLK14−. Threonine residue corresponding to the T210 in PLK1 and to T219 in PLK3 is shown in bold. ( B ) EGFP-PLK3-WT and EGFP-PLK3-T219A was immunoprecipitated from transiently transfected cells using GFP-Trap and was probed with antibody against pT210-PLK1 or GFP. ( C ) EGFP-PLK3 was isolated from cells treated with mock or calyculin using GFP Trap. Isolated EGFP-PLK3 was incubated or not with lambda phosphatase for 20 min. All samples were probed with pT210 antibody. ( D ) HEK293-EGFP-PLK3 cells were transfected with PP6C and/or PPP6R1, and PLK3 was isolated using GFP-Trap. Level of PLK3 phosphorylation was evaluated by pT210 antibody. ( E ) HEK293-EGFP-PLK3 cells were transfected with negative control siRNA or siRNA to PP6C and grown for 48 h. Where indicated, cells were treated with calyculin 20 min prior to harvesting. PLK3 was isolated with GFP Trap. Level of PLK3 phosphorylation was evaluated by pT210 antibody. Kinase assay was performed as in F. ( F ) EGFP-PLK3-WT, EGFP-PLK3-T219A, EGFP-PLK3-T219D or a kinase dead EGFP-PLK3-K91R were purified from transiently transfected 293 cells using GFP Trap and incubated with casein and 32 P-γ-ATP for 20 min at 30 °C ( n = 2). Phosphorylation was detected by autoradiography and amount of precipitated PLK3 by staining with GFP antibody. Arrowhead indicates identical position of the casein on the electrophoretic gel. ( G ) EGFP or EGFP-PLK3-WT was purified from 293 cells transfected with mock, PP6C or PP6C with PPP6R1, and kinase assay was performed as in F. Phosphorylation of PLK3 was evaluated with pT210-PLK1 antibody and phosphorylation of the casein by autoradiography ( n = 2). Arrowhead indicates an identical position on the gel.

Article Snippet: The following antibodies were used: rabbit monoclonal to PLK3 (clone D14F12, Cell Signaling Technology, #4896); rabbit polyclonal to PLK3 (Novus Biologicals, Abingdon, UK, NBP23-2530); goat polyclonal to PLK3 (Bio-Rad, Hercules, CA, USA, VPA00063); rabbit polyclonal to PLK3 (Sigma-Aldrich, St. Louis, MO, USA, HPA060318); mouse monoclonal to PLK3 (clone B37-2, BD Pharmingen, San Jose, CA, USA, 556518); rabbit polyclonal to PLK3 (St. Johns Laboratory, London, UK, STJ93099); rabbit monoclonal Phospho-PLK1 (Thr210) Antibody (Cell Signaling Technology, Danvers, MA, USA #9062); γ-tubulin (Sigma-Aldrich); GM130, p38-pT180/pY182, cJun-pSer73, phospho-histone H2AX-Ser139 (referred to as γ-H2AX) and HIF1α (Cell Signaling Technology); PPP6R1, PPP6R2 and PPP6R3 (A300-968A, A300-970A, A300-972A, Bethyl Laboratories, Montgomery, TX, USA); 14-3-3, mouse monoclonal to PPP6C and TFIIH (Santa Cruz Biotechnology, Dallas, TX, USA); and rabbit polyclonal to PPP6C (Abcam, Cambridge, UK).

Techniques: Phospho-proteomics, Residue, Immunoprecipitation, Transfection, Isolation, Incubation, Negative Control, Kinase Assay, Purification, Autoradiography, Staining