Journal: Experimental Hematology & Oncology

Article Title: Extracellular matrix stiffness reduces DNA 6 ma level to facilitate colorectal cancer progression via disrupting P53 binding to CDKN1A promoter

doi: 10.1186/s40164-025-00704-w

Figure Lengend Snippet: ALKBH1 inhibited the transcription of CDKN1A. ( A-B ) Volcano plot ( A ) and KEGG-enrichment pathways ( B ) of differentially expressed genes between shALKBH1 HCT116 cells and controls. ( C ) Volcano plot of genes related to P53 pathway RNA microarray in HCT116 cells between shALKBH1 and controls. ( D-E ) Histograms of luciferase activity of CDKN1A in HCT116 (up panel) and RKO cells (down panel) with ALKBH1 knockdown ( D ) and ALKBH1 expression ( E ). ( F-G ) Histograms of CDKN1A mRNA expression in HCT116 (up panel) and RKO cells (down panel) with ALKBH1 knockdown ( F ) and ALKBH1 expression ( G ). ( H-I ) Western blot of CDKN1A and ALKBH1 expression in HCT116 (left panel) and RKO cells (right panel) with ALKBH1 overexpression ( H ) and ALKBH1 knockdown ( I ), using GAPDH as a control. ( J ) Histograms of CDKN1A luciferase activity in HCT116 (left panel) and RKO cells (right panel) stimulated by soft and stiff substrate. ( K ) Western blot showed Flag, ALKBH1, CDKN1A and P53 expression in HCT116 cells with ALKBH1 overexpression and control after soft and stiff substrate stimulation, using GAPDH as a control. ( L ) Histogram of CTG luminescence in in HCT116 cells with ALKBH1 overexpression and control after soft and stiff substrate stimulation, n = 6 per group. ( M ) Western blot of Flag, ALKBH1 and CDKN1A expression in HCT116 cells transfecting ALKBH1 and/or CDKN1A plasmids, using GAPDH as a control. (N-O) CCK8 assays ( n = 5 per group) ( N ) and CTG luminescence histogram ( n = 6 per group) (O) of HCT116 cells transfecting ALKBH1 and/or CDKN1A plasmids. Data are represented as mean ± SEM. * P < 0.05; ns, not significant

Article Snippet: Normal rabbit IgG (#2729), Anti-CDKN1A antibody (#2947), Anti-P53 antibody (#9282), Anti-p-P53 antibody (#9284), Anti-vimentin antibody (#3932) and Anti-E-Cadherin antibody (#3195) were purchased from Cell Signaling Technology. β-Aminopropionitrile (BAPN) (HY-Y1750) was purchased from MedChemExpress.

Techniques: Microarray, Luciferase, Activity Assay, Knockdown, Expressing, Western Blot, Over Expression, Control

Journal: Experimental Hematology & Oncology

Article Title: Extracellular matrix stiffness reduces DNA 6 ma level to facilitate colorectal cancer progression via disrupting P53 binding to CDKN1A promoter

doi: 10.1186/s40164-025-00704-w

Figure Lengend Snippet: ALKBH1 attenuates P53 binding to the p21 promoter. ( A ) Spearman correlation analysis of TP53 and ALKBH1 from TCGA combing with GTEx database. ( B-C ) Histograms of P53 mRNA expression in HCT116 (up panel) and RKO cells (down panel) with ALKBH1 overexpression ( B ) and ALKBH1 knockdown ( C ). ( D ) Western blot showed ALKBH1, P-P53, and P53 expression in HCT116 (left panel) and RKO cells (right panel) with ALKBH1 overexpression (up panel) and ALKBH1 knockdown (down panel), using GAPDH as a control. ( E ) Predictive motif of p53-bound methylation-modified regions. ( F-G ) Histograms of CDKN1A luciferase activity ( F ) and CDKN1A mRNA expression ( G ) in RKO (left panel), HCT116 (middle panel), and p53-knockout HCT116 cells (right panel) overexpressing vector, wild-type ALKBH1, and mutant ALKBH1 plasmids. ( H ) Western blot of ALKBH1, P53, and CDKN1A in HCT116 and p53-knockout HCT116 cells overexpressing control, wild-type ALKBH1, and mutant ALKBH1 plasmids, using GAPDH as a control. (I) ChIP assay detected 6 mA modification levels on the CDKN1A promoter using anti-6 mA or control IgG antibody, combined with qPCR assay in HCT116 cells transfected with plasmid vector, wild-type ALKBH1 and mutant ALKBH1 plasmids. (J) ChIP assay showed binding ability of p53 to the CDKN1A promoter using anti-p53 or control IgG antibody, combined with qPCR assay in HCT116 cells transfected with plasmid vector, wild-type ALKBH1 and mutant ALKBH1 plasmids Data are represented as mean ± SEM. * P < 0.05; ns, not significant; WT, wild-type; Mut, mutant

Article Snippet: Normal rabbit IgG (#2729), Anti-CDKN1A antibody (#2947), Anti-P53 antibody (#9282), Anti-p-P53 antibody (#9284), Anti-vimentin antibody (#3932) and Anti-E-Cadherin antibody (#3195) were purchased from Cell Signaling Technology. β-Aminopropionitrile (BAPN) (HY-Y1750) was purchased from MedChemExpress.

Techniques: Binding Assay, Expressing, Over Expression, Knockdown, Western Blot, Control, Methylation, Modification, Luciferase, Activity Assay, Knock-Out, Plasmid Preparation, Mutagenesis, Transfection

Journal: Stem cell research & therapy

Article Title: Extracellular vesicles derived from human umbilical cord mesenchymal stem cells alleviate osteoarthritis of the knee in mice model by interacting with METTL3 to reduce m6A of NLRP3 in macrophage.

doi: 10.1186/s13287-022-03005-9

Figure Lengend Snippet: Fig. 2 HucMSCs-EVs alleviate knee osteoarthritis in DMM-induced OA mice model. A. Safranin O and Fast Green staining of knee sections and micro-CT analysis of knee joint in mice with DMM-induced OA. Expression levels of COL2A1, Aggrecan, ADAMTS5, and MMP13 were determined by immunofluorescence staining. Scale bar = 400 μm for Safranin O and Fast Green staining; Scale bar = 1 mm for Micro-CT; Scale bar = 100 μm for immunofluorescence staining. B. OARIS scores of knee sections in each group (n = 10). C. Osteophyte score of each group (n = 10). D. Quantification of mean fluorescence intensity of COL2A1, Aggrecan, ADAMTS5, and MMP13 in each group (n = 10). E. Levels of IL-1β, IL-18, and TNF-α in mice knee tissue as determined by ELISA (n = 3). Data are presented as mean ± SD. ns, no significance; *p < 0.05; **p < 0.01; ***p < 0.001

Article Snippet: Primary antibodies used in this experiment included antibodies against COL2A1 (1:2000, Abcam, ab188570), antibodies against Aggrecan (1:2000, Proteintech, 13,880–1-AP), antibodies against ADAMTS5 (1:2000, Abcam, ab182795), antibodies against MMP13 (1:2000, Proteintech, 18,165–1-AP), antibodies against CD9 (1:2000; Abcam; ab223052), antibodies against CD63 (1:2000; Abcam; ab134045), antibodies against Alix (1:1500; Proteintech; 12,422–1-AP), antibodies against TSG101 (1:2000; Proteintech; 14,497–1-AP), antibodies against Calnexin (1:2000; Proteintech; 10,427–2- AP), antibodies against Tubulin (1:2000; Proteintech; 10,094–1-AP), antibodies against Ago2 (1:2000; Abcam; ab186733), antibodies against NLRP3 (1:1000, Adipogen, AG-20B-0014), antibodies against METTL3 (1:2000, Proteintech; 15,073–1-AP), antibodies against IL-1β (1:1000, R&D Systems, AF-401-NA), antibodies against Ubiquitin (1:1000, Santa Cruz, sc-8017), and antibodies against caspase-1 (1:1000, Abcam, ab108362). miRNA target prediction analysis TargetScan (http:// www. targe tscan. org/ vert 80/), GeneCards (https:// www. genec ards. org/), and miRWalk (http:// mirwa lk. umm. uni- heide lberg. de/) were used to predict the miRNA of target genes of interest, and the target miRNA was determined by the intersection of prediction results. miRNA microarray assay Suri Medicine Company conducted miRNA arrays on hucMSCs-EVs (Nanjing, China).

Techniques: Staining, Micro-CT, Expressing, Immunofluorescence, Fluorescence, Enzyme-linked Immunosorbent Assay

Journal: Stem cell research & therapy

Article Title: Extracellular vesicles derived from human umbilical cord mesenchymal stem cells alleviate osteoarthritis of the knee in mice model by interacting with METTL3 to reduce m6A of NLRP3 in macrophage.

doi: 10.1186/s13287-022-03005-9

Figure Lengend Snippet: Fig. 3 The protection effect of knee OA by hucMSCs-EVs was abolished in NLRP3−/− mice. A. Safranin O and Fast Green staining of knee sections and micro-CT analysis of knee joint in NLRP3−/− mice with DMM-induced OA. Expression levels of COL2A1, Aggrecan, ADAMTS5, and MMP13 were determined by immunofluorescence staining. Scale bar = 400 μm for Safranin O and Fast Green staining; Scale bar = 1 mm for Micro-CT; Scale bar = 100 μm for immunofluorescence staining. B. OARIS scores of knee sections in each group (n = 10). C. Osteophyte score of each group (n = 10). D. Quantification of mean fluorescence intensity of COL2A1, Aggrecan, ADAMTS5, and MMP13 in each group (n = 10). E. Levels of IL-1β, IL-18, and TNF-α in mice knee tissue as determined by ELISA (n = 3). Data are presented as mean ± SD. ns, no significance; *p < 0.05; **p < 0.01; ***p < 0.001

Article Snippet: Primary antibodies used in this experiment included antibodies against COL2A1 (1:2000, Abcam, ab188570), antibodies against Aggrecan (1:2000, Proteintech, 13,880–1-AP), antibodies against ADAMTS5 (1:2000, Abcam, ab182795), antibodies against MMP13 (1:2000, Proteintech, 18,165–1-AP), antibodies against CD9 (1:2000; Abcam; ab223052), antibodies against CD63 (1:2000; Abcam; ab134045), antibodies against Alix (1:1500; Proteintech; 12,422–1-AP), antibodies against TSG101 (1:2000; Proteintech; 14,497–1-AP), antibodies against Calnexin (1:2000; Proteintech; 10,427–2- AP), antibodies against Tubulin (1:2000; Proteintech; 10,094–1-AP), antibodies against Ago2 (1:2000; Abcam; ab186733), antibodies against NLRP3 (1:1000, Adipogen, AG-20B-0014), antibodies against METTL3 (1:2000, Proteintech; 15,073–1-AP), antibodies against IL-1β (1:1000, R&D Systems, AF-401-NA), antibodies against Ubiquitin (1:1000, Santa Cruz, sc-8017), and antibodies against caspase-1 (1:1000, Abcam, ab108362). miRNA target prediction analysis TargetScan (http:// www. targe tscan. org/ vert 80/), GeneCards (https:// www. genec ards. org/), and miRWalk (http:// mirwa lk. umm. uni- heide lberg. de/) were used to predict the miRNA of target genes of interest, and the target miRNA was determined by the intersection of prediction results. miRNA microarray assay Suri Medicine Company conducted miRNA arrays on hucMSCs-EVs (Nanjing, China).

Techniques: Staining, Micro-CT, Expressing, Immunofluorescence, Fluorescence, Enzyme-linked Immunosorbent Assay

Journal: Stem cell research & therapy

Article Title: Extracellular vesicles derived from human umbilical cord mesenchymal stem cells alleviate osteoarthritis of the knee in mice model by interacting with METTL3 to reduce m6A of NLRP3 in macrophage.

doi: 10.1186/s13287-022-03005-9

Figure Lengend Snippet: Fig. 8 miR-1208 alleviates knee osteoarthritis and decreases the cartilage degradation in vivo. A. Safranin O and Fast Green, Micro-CT, and immunofluorescence staining of each group of mice knee section. B. OARIS scores of knee sections in each group (n = 10). C. Osteophyte score of each group (n = 10). D. Quantification of mean fluorescence intensity of COL2A1, Aggrecan, ADAMTS5, and MMP13 in each group (n = 10)

Article Snippet: Primary antibodies used in this experiment included antibodies against COL2A1 (1:2000, Abcam, ab188570), antibodies against Aggrecan (1:2000, Proteintech, 13,880–1-AP), antibodies against ADAMTS5 (1:2000, Abcam, ab182795), antibodies against MMP13 (1:2000, Proteintech, 18,165–1-AP), antibodies against CD9 (1:2000; Abcam; ab223052), antibodies against CD63 (1:2000; Abcam; ab134045), antibodies against Alix (1:1500; Proteintech; 12,422–1-AP), antibodies against TSG101 (1:2000; Proteintech; 14,497–1-AP), antibodies against Calnexin (1:2000; Proteintech; 10,427–2- AP), antibodies against Tubulin (1:2000; Proteintech; 10,094–1-AP), antibodies against Ago2 (1:2000; Abcam; ab186733), antibodies against NLRP3 (1:1000, Adipogen, AG-20B-0014), antibodies against METTL3 (1:2000, Proteintech; 15,073–1-AP), antibodies against IL-1β (1:1000, R&D Systems, AF-401-NA), antibodies against Ubiquitin (1:1000, Santa Cruz, sc-8017), and antibodies against caspase-1 (1:1000, Abcam, ab108362). miRNA target prediction analysis TargetScan (http:// www. targe tscan. org/ vert 80/), GeneCards (https:// www. genec ards. org/), and miRWalk (http:// mirwa lk. umm. uni- heide lberg. de/) were used to predict the miRNA of target genes of interest, and the target miRNA was determined by the intersection of prediction results. miRNA microarray assay Suri Medicine Company conducted miRNA arrays on hucMSCs-EVs (Nanjing, China).

Techniques: In Vivo, Micro-CT, Immunofluorescence, Staining, Fluorescence

Journal: eLife

Article Title: Mechanically transduced immunosorbent assay to measure protein-protein interactions

doi: 10.7554/eLife.67525

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-GST (rabbit polyclonal) , Epicypher , 13–0022 , (1:1000).

Techniques: Recombinant, Plasmid Preparation, Software, Avidin-Biotin Assay, Peptide Microarray

Journal: eLife

Article Title: GIV/Girdin, a non-receptor modulator for Gαi/s, regulates spatiotemporal signaling during sperm capacitation and is required for male fertility

doi: 10.7554/elife.69160

Figure Lengend Snippet: Figure 1. GIV (CCDC88A) is highly expressed in spermatocytes in testis and localizes to the acrosomal cap. (A) Bar graph displays the relative fluorescence unit (RFU) of endogenous full-length GIV protein in immunoblots of organ lysates published previously using three independent anti-GIV antibodies raised against different epitopes of GIV (Anai et al., 2005). (Figure 1—source data 1)(B) RNA expression in the single-cell-type clusters identified in the human testis visualized by a UMAP plot (inset) and a bar plot. The bar plot shows RNA expression (pTPM) in each cell-type cluster.

Article Snippet: Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody Rabbit polyclonal anti- GIV (Girdin) (T- 13) Santa Cruz Biotechnology sc- 133371 Antibody Rabbit monoclonal diagnostic grade anti- Girdin/GIV antibody Custom; Sprint Bioscience SP173 Validated in prior publication Ghosh et al., 2016 Antibody Rabbit polyclonal anti- GIV (Girdin) (CC- Ab) Millipore Sigma ABT80 Antibody Rabbit polyclonal anti- GIV pS1675 Ab Custom, from 21t Century Biosciences n/a Validated in prior publication Bhandari et al., 2015 Antibody Rabbit polyclonal anti- GIV pS1689 Ab Custom, from 21st Century Biosciences n/a Validated in prior publication LópezSánchez et al., 2013 Antibody Rabbit monoclonal anti- GIV pY1764 Ab Custom, Spring Biosciences Inc n/a Validated in prior publications Midde et al., 2015; Lin et al., 2011; Midde et al., 2018; Dunkel et al., 2016 Antibody Rabbit monoclonal antipT308 AKT Cell Signaling Technology D9E Antibody Mouse monoclonal anti- total AKT Cell Signaling Technology 40D4 Antibody Mouse anti- sp56 Thermo Fisher Scientific (Waltham, MA) MA1- 10866 Antibody Mouse anti- human hexokinase 1/2 monoclonal antibody R&D Systems, (Minneapolis, MN) MAB8179 Antibody Rabbit anti- phospho- PKA substrate (RRXS*/T*)100G7E Cell Signaling Technology 9624 Antibody Goat anti- rabbit IgG, Alexa Fluor 594 conjugated ThermoFisher Scientific A11072 For immunofluorescence (IF) Antibody Goat anti- mouse IgG, Alexa Fluor 488 conjugated ThermoFisher Scientific A11017 For immunofluorescence (IF) Antibody IRDye 800CW goat antimouse IgG secondary (1:10,000) LI- COR Biosciences 926- 32210 For immunoblotting Antibody IRDye 680RD goat anti- rabbit IgG secondary (1:10,000) LI- COR Biosciences 926- 68071 For immunoblotting Reynoso, Castillo, Katkar et al. eLife 2021;10:e69160.

Techniques: Fluorescence, Western Blot, RNA Expression

Journal: eLife

Article Title: GIV/Girdin, a non-receptor modulator for Gαi/s, regulates spatiotemporal signaling during sperm capacitation and is required for male fertility

doi: 10.7554/elife.69160

Figure Lengend Snippet: Figure 2. Transcripts of CCDC88A (GIV) are downregulated in infertile male testis and semen. (A) Schematic displays the approach used to search NCBI GEO database for testis and sperm transcriptomic datasets suitable to study correlations between the abundance of CCDC88A transcripts and male fertility. (B–E) Whisker plots show the relative abundance of CCDC88A (expressed as Log2 normalized expression; see Materials and methods for different normalization approaches used for microarray and RNA-seq datasets) in sperm or testis samples (as annotated using schematics) in samples

Article Snippet: Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody Rabbit polyclonal anti- GIV (Girdin) (T- 13) Santa Cruz Biotechnology sc- 133371 Antibody Rabbit monoclonal diagnostic grade anti- Girdin/GIV antibody Custom; Sprint Bioscience SP173 Validated in prior publication Ghosh et al., 2016 Antibody Rabbit polyclonal anti- GIV (Girdin) (CC- Ab) Millipore Sigma ABT80 Antibody Rabbit polyclonal anti- GIV pS1675 Ab Custom, from 21t Century Biosciences n/a Validated in prior publication Bhandari et al., 2015 Antibody Rabbit polyclonal anti- GIV pS1689 Ab Custom, from 21st Century Biosciences n/a Validated in prior publication LópezSánchez et al., 2013 Antibody Rabbit monoclonal anti- GIV pY1764 Ab Custom, Spring Biosciences Inc n/a Validated in prior publications Midde et al., 2015; Lin et al., 2011; Midde et al., 2018; Dunkel et al., 2016 Antibody Rabbit monoclonal antipT308 AKT Cell Signaling Technology D9E Antibody Mouse monoclonal anti- total AKT Cell Signaling Technology 40D4 Antibody Mouse anti- sp56 Thermo Fisher Scientific (Waltham, MA) MA1- 10866 Antibody Mouse anti- human hexokinase 1/2 monoclonal antibody R&D Systems, (Minneapolis, MN) MAB8179 Antibody Rabbit anti- phospho- PKA substrate (RRXS*/T*)100G7E Cell Signaling Technology 9624 Antibody Goat anti- rabbit IgG, Alexa Fluor 594 conjugated ThermoFisher Scientific A11072 For immunofluorescence (IF) Antibody Goat anti- mouse IgG, Alexa Fluor 488 conjugated ThermoFisher Scientific A11017 For immunofluorescence (IF) Antibody IRDye 800CW goat antimouse IgG secondary (1:10,000) LI- COR Biosciences 926- 32210 For immunoblotting Antibody IRDye 680RD goat anti- rabbit IgG secondary (1:10,000) LI- COR Biosciences 926- 68071 For immunoblotting Reynoso, Castillo, Katkar et al. eLife 2021;10:e69160.

Techniques: Whisker Assay, Expressing, Microarray, RNA Sequencing

Journal: eLife

Article Title: GIV/Girdin, a non-receptor modulator for Gαi/s, regulates spatiotemporal signaling during sperm capacitation and is required for male fertility

doi: 10.7554/elife.69160

Figure Lengend Snippet: Figure 8. Summary and working model: spatiotemporally segregated roles of GIV/Girdin during sperm capacitation. Schematic summarizes the key findings in this work and places them in the context of existing literature. GIV is likely to primarily function during capacitation of sperm, during which it fulfills two key roles as a signal transducer in a spatiotemporally segregated manner. The first role (right, top) is in the head of the sperm, where GIV’s GEM motif inhibits the AC→cAMP pathway and prevents acrosomal reaction. The second role (right, bottom) is in the mid-piece and tail region of the sperm, which involves tyrosine phosphorylation of GIV, which

Article Snippet: Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody Rabbit polyclonal anti- GIV (Girdin) (T- 13) Santa Cruz Biotechnology sc- 133371 Antibody Rabbit monoclonal diagnostic grade anti- Girdin/GIV antibody Custom; Sprint Bioscience SP173 Validated in prior publication Ghosh et al., 2016 Antibody Rabbit polyclonal anti- GIV (Girdin) (CC- Ab) Millipore Sigma ABT80 Antibody Rabbit polyclonal anti- GIV pS1675 Ab Custom, from 21t Century Biosciences n/a Validated in prior publication Bhandari et al., 2015 Antibody Rabbit polyclonal anti- GIV pS1689 Ab Custom, from 21st Century Biosciences n/a Validated in prior publication LópezSánchez et al., 2013 Antibody Rabbit monoclonal anti- GIV pY1764 Ab Custom, Spring Biosciences Inc n/a Validated in prior publications Midde et al., 2015; Lin et al., 2011; Midde et al., 2018; Dunkel et al., 2016 Antibody Rabbit monoclonal antipT308 AKT Cell Signaling Technology D9E Antibody Mouse monoclonal anti- total AKT Cell Signaling Technology 40D4 Antibody Mouse anti- sp56 Thermo Fisher Scientific (Waltham, MA) MA1- 10866 Antibody Mouse anti- human hexokinase 1/2 monoclonal antibody R&D Systems, (Minneapolis, MN) MAB8179 Antibody Rabbit anti- phospho- PKA substrate (RRXS*/T*)100G7E Cell Signaling Technology 9624 Antibody Goat anti- rabbit IgG, Alexa Fluor 594 conjugated ThermoFisher Scientific A11072 For immunofluorescence (IF) Antibody Goat anti- mouse IgG, Alexa Fluor 488 conjugated ThermoFisher Scientific A11017 For immunofluorescence (IF) Antibody IRDye 800CW goat antimouse IgG secondary (1:10,000) LI- COR Biosciences 926- 32210 For immunoblotting Antibody IRDye 680RD goat anti- rabbit IgG secondary (1:10,000) LI- COR Biosciences 926- 68071 For immunoblotting Reynoso, Castillo, Katkar et al. eLife 2021;10:e69160.

Techniques: Phospho-proteomics

Journal: Age

Article Title: Global gene expression profile of normal and regenerating liver in young and old mice

doi: 10.1007/s11357-015-9796-7

Figure Lengend Snippet: Expression of cell cycle genes in old vs young mice after PH. Livers of hepatectomized young or old mice were analyzed at indicated time points

Article Snippet: The following antibodies were used: mouse monoclonal antibodies against cyclin D1 (72–13), cyclin D2, cyclin D3, and proliferating cell nuclear antigen (PCNA) (Santa Cruz Biotechnology, CA); actin (clone AC-40) (Sigma Chem.

Techniques: Expressing, Transformation Assay, Derivative Assay

Journal: Age

Article Title: Global gene expression profile of normal and regenerating liver in young and old mice

doi: 10.1007/s11357-015-9796-7

Figure Lengend Snippet: YAP expression in old and young livers after PH. a Microarray analysis of Yap mRNA. mRNA expression was determined in old and young mice after PH. Gene expression is reported as fold difference relative to age-matched controls. b Western blot analysis of YAP and phospho-YAP proteins. Old and young mice were sacrificed 12, 24, and 36 h after 2/3 PH. Total extracts (100 to 150 mg/lane) were prepared as described in “Materials and methods” section. Appropriate loading was confirmed by staining the gel with Coomassie Blue and using anti-actin antibody as loading control. Each lane represents a pool of three livers. CO controls. c Western blot analysis of cyclin D1, PCNA, YAP, and phospho-YAP in livers from mice subjected to PH with or without TCPBOP pretreatment (TCP + PH and PH, respectively). Young mice were subjected to PH 3 days after treatment with the mitogen TCP, as described in “Materials and methods” section. Animals were sacrificed 72 h after PH. Total extracts (100 to 150 mg/lane) were prepared from the livers, and Western analysis was performed as described in “Materials and methods” section. Appropriate loading was confirmed by staining the gel with Coomassie Blue and using anti-actin antibody as loading control. Each lane represents a pool of three livers. CO controls. d Western blot analysis of nuclear YAP in TCP + PH or PH livers. Nuclear extracts for YAP (100 to 150 mg/lane) were prepared, and Western analysis was performed as described in “Materials and methods” section. Appropriate loading was confirmed by staining the gel with Coomassie Blue and using anti-albumin antibody as loading control. Each lane represents a pool of three livers. CO controls

Article Snippet: The following antibodies were used: mouse monoclonal antibodies against cyclin D1 (72–13), cyclin D2, cyclin D3, and proliferating cell nuclear antigen (PCNA) (Santa Cruz Biotechnology, CA); actin (clone AC-40) (Sigma Chem.

Techniques: Expressing, Microarray, Gene Expression, Western Blot, Staining, Control

Journal: Epigenetics & Chromatin

Article Title: The histone and non-histone methyllysine reader activities of the UHRF1 tandem Tudor domain are dispensable for the propagation of aberrant DNA methylation patterning in cancer cells

doi: 10.1186/s13072-020-00366-4

Figure Lengend Snippet: LIG1K126me2 is read by a high-affinity interaction through the UHRF1 TTD. a Protein reader domain microarrays consisting of 308 GST-tagged domains (see Additional file : Fig. S1), each printed in duplicate, were probed by either anti-GST antibody, or Cy3-labeled LIG1 (118–130) K126me2, H3 (1–20) K9me2, or LIG1 (118–130) K126me0 (left). Reader arrays were quantified using ArrayNinja software. Data were normalized to the brightest signal for each peptide (right). Error bars represent the range from duplicate spots. Full reader array datasets are available in Additional file : Table S1. b The indicated GST-tagged reader domains were hybridized to histone peptide microarrays at 1 µM, followed by fluorescent detection and quantified by ArrayNinja. Results for interactions with H3 (1–20) K9me2 and LIG1 (118–130) K126me2 are shown. Error bars represent standard error of the mean of six printed spots. Full histone peptide array data are available in Additional file : Table S2. c Fluorescence polarization binding assays between the indicated GST-tagged reader domains and either FAM-LIG1 (118–130) K126me2 (left) or H3 (1–20) K9me2-FAM (right). Error bars represent the 95% confidence interval from triplicate measurements

Article Snippet: Antibodies and dilutions used were as follows: LIG1 (ProteinTech 18051–1-AP, 1:1000, lot # unk.), UHRF1 (Cell Signaling 12387, 1:1000, lot # 1), FLAG (Sigma 1804, 1:5000, lot # unk), beta 3 Tubulin (UniProtKB Q13509, ProteinTech 66240-1-Ig, 1:100,000, lot # unk.), histone H3 (EpiCypher 13–0001, 1:100,000, lot # 12320001), H3K9me2 (Abcam 1220, 1:5000, lot # unk.), and H3K9me3 (Active Motif 39161, 1:5000, lot # unk).

Techniques: Labeling, Software, Peptide Microarray, Fluorescence, Binding Assay

Journal: Epigenetics & Chromatin

Article Title: The histone and non-histone methyllysine reader activities of the UHRF1 tandem Tudor domain are dispensable for the propagation of aberrant DNA methylation patterning in cancer cells

doi: 10.1186/s13072-020-00366-4

Figure Lengend Snippet: Unlike binding to H3K9me2, the UHRF1 PHD and its N-terminal linker do not modulate LIG1K126me2 recognition through the TTD. a Structural models of UHRF1 TTD bound to LIG1K126me3 (PDB:5YYA), UHRF1 PBR (PDB:6B9M), H3K9me3 (PDB:2L3R), and UHRF1 TTD–PHD bound to H3K9me3 (PDB:3ASK) with the linker between TTD and PHD shown in pink. b , c FP binding assays between the indicated GST-tagged b UHRF1 domains or c TTD–PHD mutants reader domains and FAM-LIG1 (118–130) K126me2, H3 (1–20) K9me2-FAM, or FAM-H3 (1–20) K9me2. Error bars represent standard error of the mean from triplicate measurements

Article Snippet: Antibodies and dilutions used were as follows: LIG1 (ProteinTech 18051–1-AP, 1:1000, lot # unk.), UHRF1 (Cell Signaling 12387, 1:1000, lot # 1), FLAG (Sigma 1804, 1:5000, lot # unk), beta 3 Tubulin (UniProtKB Q13509, ProteinTech 66240-1-Ig, 1:100,000, lot # unk.), histone H3 (EpiCypher 13–0001, 1:100,000, lot # 12320001), H3K9me2 (Abcam 1220, 1:5000, lot # unk.), and H3K9me3 (Active Motif 39161, 1:5000, lot # unk).

Techniques: Binding Assay

Journal: Nature Communications

Article Title: Increased translation driven by non-canonical EZH2 creates a synthetic vulnerability in enzalutamide-resistant prostate cancer

doi: 10.1038/s41467-024-53874-2

Figure Lengend Snippet: a Heatmap of RNAseq data from the International SU2C/PCF Dream Team metastatic CRPC dataset with the inclusion of only the top PRKCI -low (n = 30) and PRKCI -high (n = 30) samples shown. b Representative images of immunofluorescence staining for EZH2, PKCλ/ι, and DAPI in prostates from TRAMP + mice (n = 3 mice). Scale bars 100 μm. c Representative images of immunofluorescence staining for EZH2, PKCλ/ι, and DAPI in prostate tumors from Pten f/f Rb1 f/f MYCN + PbCre + mice (n = 3 mice per group). Scale bars, 100 μm. d , e Quantification of nuclear EZH2 staining in CRPC samples (n = 177) from a tissue microarray (WCM TMA). CRPC patients were categorized in high or low PKCλ/ι ( d ). Representative images of immunofluorescence staining for EZH2, PKCλ/ι, and DAPI in CRPC samples from the TMA (n = 177). Scale bars, 100 μm ( e ). f Heatmap of RNA expression for neuroendocrine (NE)-related genes of human prostate organoids from GSE181374 . g Immunoblots in human adenocarcinoma organoids (MSKPCa3) and NEPC organoids (WCM1262, WCM1078, and WCM154), and quantification (n = 3 independent experiments). h Representative images of immunofluorescence staining for EZH2, and DAPI in prostate tumors from Pten f/f PbCre + and Pten f/f Prkci f/f PbCre + mice (n = 3 mice per group). Scale bars, 100 μm. i Immunoblots in sg PRKCI and sgC LNCaP cells (n = 3 independent experiments). j Immunofluorescence staining of EZH2 in sg PRKCI and sgC LNCaP cells and quantification of EZH2 intensity (sgC: n = 181, sg PRKCI : n = 139 cells examined). Scale bars, 20 μm. k Representative images of immunofluorescence staining for EZH2, and Phalloidin in Pten Δ/Δ and Pten Δ/Δ Prkci Δ/Δ prostate organoids (n = 3 biological replicates). Scale bars, 20 μm. l Immunoblots in nuclear fraction from Pten Δ/Δ , Pten Δ/Δ Prkci Δ/Δ , Pten Δ/Δ Rb1 Δ/Δ , and Pten Δ/Δ Rb1 Δ/Δ Prkci Δ/Δ prostate organoids, and quantification (n = 3 independent experiments). Data shown as mean ± SEM ( g , j , l ). Pearson correlation of pairwise comparisons with PRKCI ( a ). Two-tailed Chi-square test ( d ). Two-tailed unpaired Student’s t-test ( g , j , l ). Source data are provided as a file.

Article Snippet: The following antibodies were used: CUTANA™ IgG Negative Control Antibody for CUT&RUN and CUT&Tag (#13-0042, 1:50), rabbit anti-H3K4me3 SNAP-Certified™ for CUT&RUN (#13-0041, 1:50), rabbit anti-EZH2 CUTANA™ CUT&RUN (#13-2026, 1:50), rabbit anti-H3K27me3 SNAP-Certified™ for CUT&RUN and CUT&Tag (#13-0055, 1:50) from Epicypher and rabbit anti-YY1 (#46395, 1:50) from Cell Signaling Technology.

Techniques: Immunofluorescence, Staining, Microarray, RNA Expression, Western Blot, Two Tailed Test

Journal: Nature Communications

Article Title: Increased translation driven by non-canonical EZH2 creates a synthetic vulnerability in enzalutamide-resistant prostate cancer

doi: 10.1038/s41467-024-53874-2

Figure Lengend Snippet: a Immunoblots in HEK293T cells transfected with the indicated plasmids and quantification of HA-EZH2 (n = 3 independent experiments). b Immunoblots in LNCaP cells transfected with the indicated plasmids and treated with cycloheximide (CHX) (50 μg/ml) and MG132 (10 μM), Bafilomycin A1 (100 nM) or MLN4924 (1 μM) for 12 h, and quantification of HA-EZH2 (n = 3 independent experiments). c sg PRKCI and sgC LNCaP cells were incubated with 50 μg/ml of CHX at indicated time points, and quantification of EZH2 (n = 3 independent experiments). d Immunoblotting of EZH2 immunoprecipitates in sg PRKCI and sgC LNCaP cells, transfected with the indicated plasmids (n = 2 independent experiments). e Signal Intensity of EZH2-RBBP6 interaction measured by Mass Spectrometry in sgC and sg PRKCI HEK293T cells transfected with HA-EZH2 (n = 1 sample per condition). f Immunoblotting of HA-tagged immunoprecipitates of sg PRKCI and sgC LNCaP cells, transfected with the indicated plasmids (n = 2 independent experiments). g Immunoblotting of EZH2 immunoprecipitates in sg PRKCI and sgC LNCaP cells (n = 2 independent experiments). h Immunoblots in HEK293T cells, transfected with the indicated plasmids and quantification of EZH2 (n = 3 independent experiments). i Immunoblots in LNCaP cells, transduced with siRNAs and quantification of EZH2 (n = 3 independent experiments). j Immunofluorescence staining of EZH2 in LNCaP cells transduced with siRNAs and quantification of the EZH2 intensity (siC: n = 143, si RBBP6 : n = 138 cells examined). Scale bars 10 μm. k Immunoblots in sg PRKCI and sgC LNCaP cells, transduced with the indicated siRNAs and quantification of EZH2 (n = 3 independent experiments). l Immunoblotting of EZH2 immunoprecipitates in LNCaP cells, transduced with the indicated siRNAs (n = 2 independent experiments). m LNCaP cells, transduced with the indicated siRNAs, were treated as in ( c ), and EZH2 quantification (n = 3 independent experiments). Immunoblot experiments were performed at least two times independently, with similar results. Data shown as mean ± SEM of 3 biological replicates ( a , b , c , h , i , k , m ). Two-tailed unpaired Student’s t-test ( a , b , h , i , j , k ). Two-way ANOVA ( c , m ). Source data are provided as a file.

Article Snippet: The following antibodies were used: CUTANA™ IgG Negative Control Antibody for CUT&RUN and CUT&Tag (#13-0042, 1:50), rabbit anti-H3K4me3 SNAP-Certified™ for CUT&RUN (#13-0041, 1:50), rabbit anti-EZH2 CUTANA™ CUT&RUN (#13-2026, 1:50), rabbit anti-H3K27me3 SNAP-Certified™ for CUT&RUN and CUT&Tag (#13-0055, 1:50) from Epicypher and rabbit anti-YY1 (#46395, 1:50) from Cell Signaling Technology.

Techniques: Western Blot, Transfection, Incubation, Mass Spectrometry, Transduction, Immunofluorescence, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: Increased translation driven by non-canonical EZH2 creates a synthetic vulnerability in enzalutamide-resistant prostate cancer

doi: 10.1038/s41467-024-53874-2

Figure Lengend Snippet: a In vitro phosphorylation of HA-tagged EZH2 by recombinant PKCλ/ι (n = 3 independent experiments). b Identification of EZH2 phosphorylation sites by PKCλ/ι: HA-EZH2, in vitro phosphorylated with recombinant PKCλ/ι, or HA-EZH2 transfected into sgC and sg PRKCI cells were analyzed by MS (n = 1 sample per condition). c In vitro phosphorylation of HA-EZH2 WT or EZH2 S375/380AA as in ( a ) (n = 2 independent experiments). d Alignment of the amino acid sequence of human EZH2 (372-383 aa) with orthologs in other species. e EZH2 WT or EZH2 S375/380AA LNCaP cells were incubated with CHX (50 μg/ml) at indicated time points, and EZH2 levels were quantified (n = 3 independent experiments). f Immunofluorescent staining of EZH2 in EZH2 WT or EZH2 S375/380AA LNCaP cells and quantification (EZH2 WT : n = 91, EZH2 S375/380AA : n = 83 cells examined). Scale bars 10 μm. g , h Immunoblots of HA-tagged immunoprecipitates in HEK293T, transfected for the indicated plasmids (n = 2 independent experiments). i Immunoblots in nuclear lysates from sg PRKCI and sgC LNCaP cells (n = 2 independent experiments). j Immunofluorescence staining of pEZH2(S380) in sg PRKCI and sgC LNCaP cells (sgC: n = 459, sg PRKCI : n = 270 cells examined), and quantification. Scale bars 10 μm. k Immunoblots in Pten Δ/Δ , Pten Δ/Δ Prkci Δ/Δ , Pten Δ/Δ Rb1 Δ/Δ , and Pten Δ/Δ Rb1 Δ/Δ Prkci Δ/Δ prostate organoids (n = 3 independent experiments). l Immunofluorescence staining for pEZH2(S380), EZH2, PKCλ/ι, SYP and DAPI in prostate tumors from the NEPC model Pten f/f Rb1 f/f MYCN + PbCre + (n = 3 mice per group). Scale bars 200 μm and 20 μm. m Immunofluorescence staining for pEZH2(S380), EZH2, PKCλ/ι, and DAPI in human NEPC PDOs WCM154 (n = 1). Scale bars, 100 μm and 20 μm. n Immunoblots in human adenocarcinoma (MSKPCa3) and NEPC (WCM1262, WCM1078, WCM154) organoids (n = 3 independent experiments). o Immunoblots in control and PRKCI -overexpressed (OE) NEPC PDOs WCM1078 and WCM154 (n = 2 independent experiments). Immunoblot experiments were performed at least two times independently, with similar results. Data shown as mean ± SEM of the biological replicates ( e ). Two-way ANOVA ( e ). Two-tailed unpaired Student’s t-test ( f , j ). Source data are provided as a file.

Article Snippet: The following antibodies were used: CUTANA™ IgG Negative Control Antibody for CUT&RUN and CUT&Tag (#13-0042, 1:50), rabbit anti-H3K4me3 SNAP-Certified™ for CUT&RUN (#13-0041, 1:50), rabbit anti-EZH2 CUTANA™ CUT&RUN (#13-2026, 1:50), rabbit anti-H3K27me3 SNAP-Certified™ for CUT&RUN and CUT&Tag (#13-0055, 1:50) from Epicypher and rabbit anti-YY1 (#46395, 1:50) from Cell Signaling Technology.

Techniques: In Vitro, Recombinant, Transfection, Sequencing, Incubation, Staining, Western Blot, Immunofluorescence, Control, Two Tailed Test

Journal: Nature Communications

Article Title: Increased translation driven by non-canonical EZH2 creates a synthetic vulnerability in enzalutamide-resistant prostate cancer

doi: 10.1038/s41467-024-53874-2

Figure Lengend Snippet: a , b Dose-response curves using CFU assay for 14 days to determine the IC 50 of ENZA for sgC and sg PRKCI ( a ) or EZH2 WT , EZH2 S375/380AA ( b ) LNCaP cells. IC 50 value as the average of two biological replicates. c Dose-response curves using CFU assay for 14 days to determine the IC 50 of ENZA treated with vehicle or 5 μM GSK126 in sgC and sg PRKCI LNCaP cells. IC 50 value as the average of two biological replicates. d sgC and sg PRKCI LNCaP cells were treated with ENZA and GSK126 alone or combined and drug synergism was assessed using Bliss Independence method (n = 3 technical replicates). The positive drug synergy is represented as red peaks and the Bliss synergy scores are indicated on the 3-D plots. e Growth curves of sgC and sg PRKCI LNCaP cells treated with 5 μM ENZA and 5 μM GSK126 alone or combined. Representative experiment of two biological replicates. f , g Growth curves of Pten Δ/Δ and Pten Δ/Δ Prkci Δ/Δ ( f ) and Pten Δ/Δ Rb1 Δ/Δ and Pten Δ/Δ Rb1 Δ/Δ Prkci Δ/Δ ( g ) mouse prostate organoids treated with 5 μM ENZA and 10 μM GSK126 alone or combined. Representative experiment of two biological replicates. h Dose-response curves using CFU assay for 14 days to determine the IC 50 of ENZA treated with vehicle or 75 nM EPZ6438 in sgC and sg PRKCI LNCaP cells. IC 50 value as the average of two biological replicates. i Growth curves of WCM1262 and WCM1078 human prostate organoids treated with 10 μM ENZA and 10 μM EPZ6438 alone or combined. Representative experiment of two biological replicates. j Dose-response curves using CFU assay for 14 days to determine the IC 50 of ENZA treated with vehicle or 1 μM MS1943 in sgC and sg PRKCI LNCaP cells. IC 50 value is the average of two biological replicates. k Growth curves of Pten Δ/Δ and Pten Δ/Δ Prkci Δ/Δ mouse organoids treated with 5 μM ENZA and 5 μM MS1943 alone or combined. Representative experiment of two biological replicates. Data shown as mean ± SD of technical triplicates ( e , f , g , i , k ). Bliss synergy scores were calculated using SynergyFinder 2.2 ( d ). Two-way ANOVA ( e , f , g , i , k ). Source data are provided as a file.

Article Snippet: The following antibodies were used: CUTANA™ IgG Negative Control Antibody for CUT&RUN and CUT&Tag (#13-0042, 1:50), rabbit anti-H3K4me3 SNAP-Certified™ for CUT&RUN (#13-0041, 1:50), rabbit anti-EZH2 CUTANA™ CUT&RUN (#13-2026, 1:50), rabbit anti-H3K27me3 SNAP-Certified™ for CUT&RUN and CUT&Tag (#13-0055, 1:50) from Epicypher and rabbit anti-YY1 (#46395, 1:50) from Cell Signaling Technology.

Techniques: Colony-forming Unit Assay

Journal: Nature Communications

Article Title: Increased translation driven by non-canonical EZH2 creates a synthetic vulnerability in enzalutamide-resistant prostate cancer

doi: 10.1038/s41467-024-53874-2

Figure Lengend Snippet: a Heatmaps (CUT&RUN) for EZH2, H3K27me3 and H3K4me3 ± 8 kb from the centers of canonical EZH2 + /H3K27me3 + peaks (EZH2 ensemble; top panels) or non-canonical EZH2 + /H3K27me - /H3K4me3 + peaks (EZH2 solo; bottom panels) in sg PRKCI and sgC LNCaP cells treated or not with 10 μM ENZA for 72 h (n = 3 biological replicates). b Percentage of EZH2 solo peaks and ensemble peaks found in ( a ) (n = 3 biological replicates). c Pie-chart plot showing the genomic distribution of peaks for EZH2 ensemble or solo in sg PRKCI and sgC LNCaP cells, treated as in ( a ) (n = 3 biological replicates). d Immunoblotting of nuclear lysates and EZH2 immunoprecipitates of sgC and sg PRKCI LNCaP cells, treated as in ( a ) (n = 2 independent experiments). e – g Proximity Ligation Assay (PLA) of EZH2-EED or EZH2-SUZ12 in sg PRKCI (EZH2-EED: n = 25; EZH2-SUZ12: 40 cells examined) and sgC (EZH2-EED: n = 33; EZH2-SUZ12: 40 cells examined) LNCaP cells ( e ), Pten Δ/Δ (EZH2-EED: n = 39; EZH2-SUZ12: 39 cells examined) and Pten Δ/Δ Prkci Δ/Δ (EZH2-EED: n = 39; EZH2-SUZ12: 34 cells examined) mouse organoids ( f ), or PRKCI -OE (EZH2-EED: n = 82; EZH2-SUZ12: 75 cells examined) and Control (EZH2-EED: n = 68; EZH2-SUZ12: 75 cells examined) NEPC PD)Os WCM154 ( g ) treated as in ( a ), and quantification. Scale bars, 10 μm. h Enrichment of differential transcription factor motifs between EZH2 ensemble peaks (all genomic regions) in sg PRKCI and sgC LNCaP cells, plotted by ranks generated from their associated p values (n = 3 biological replicates). i Top 15 GO pathways from findGO.pl, analysis of genes with unique EZH2 ensemble peaks in sg PRKCI and sgC LNCaP cells treated with ENZA (n = 3 biological replicates). j Averaged signal intensities and Heatmap (CUT&RUN) for EZH2 ± 4 kb from the centers of EZH2 ensemble peaks in genes from neuronal-related pathways (n = 3 biological replicates). Immunoblot experiments were performed at least two times independently with similar results. Data shown as mean ± SEM ( e – g ). Fisher’s exact test ( b ). Two-tailed unpaired Student’s t-test ( e – g ). Source data are provided as a file.

Article Snippet: The following antibodies were used: CUTANA™ IgG Negative Control Antibody for CUT&RUN and CUT&Tag (#13-0042, 1:50), rabbit anti-H3K4me3 SNAP-Certified™ for CUT&RUN (#13-0041, 1:50), rabbit anti-EZH2 CUTANA™ CUT&RUN (#13-2026, 1:50), rabbit anti-H3K27me3 SNAP-Certified™ for CUT&RUN and CUT&Tag (#13-0055, 1:50) from Epicypher and rabbit anti-YY1 (#46395, 1:50) from Cell Signaling Technology.

Techniques: Western Blot, Proximity Ligation Assay, Control, Generated, Two Tailed Test

Journal: Nature Communications

Article Title: Increased translation driven by non-canonical EZH2 creates a synthetic vulnerability in enzalutamide-resistant prostate cancer

doi: 10.1038/s41467-024-53874-2

Figure Lengend Snippet: a Enrichment of differential transcription factor motifs between EZH2 solo peaks (all genomic regions) in sg PRKCI and sgC LNCaP cells, plotted by ranks generated from their associated p values (n = 3 biological replicates). b Heatmap of CUT&RUN for YY1 ± 8 kb from the centers of EZH2 solo or ensemble peaks in sg PRKCI and sgC LNCaP cells treated with 10 μM ENZA for 72 h (n = 3 biological replicates). c , Venn diagrams showing the overlap of EZH2 solo or ensemble peaks with YY1 in sg PRKCI and sgC LNCaP cells treated as in ( b ) (n = 3 biological replicates). d , e Immunoblots and quantification of soluble EZH2 ( d ) or YY1 ( e ) extracted using sequential salt extraction assay from sgC and sg PRKCI LNCaP cells treated or not with ENZA (n = 3 independent experiments). f Immunoblots of EZH2 immunoprecipitates in LNCaP cells treated or not with 10 μM ENZA for 72 h (n = 2 independent experiments). g PLA of EZH2 and YY1 in sgC and sg PRKCI LNCaP cells treated or not with 10 μM GSK126 and 10 μM ENZA for 72 h and quantification (sgC-DMSO: n = 40, sg PRKCI -DMSO: n = 41, sgC-GSK126: n = 43, sg PRKCI -GSK126: n = 42 cells examined). Scale bars, 10 μm. h , i PLA of EZH2 and YY1 in Pten Δ/Δ and Pten Δ/Δ Prkci Δ/Δ , Pten Δ/Δ Rb1 Δ/Δ and Pten Δ/Δ Rb1 Δ/Δ Prkci Δ/Δ mouse prostate organoids ( h ), or PRKCI- overexpressing ( PRKCI -OE) and control NEPC PDOs WCM154 ( i ), with quantification ( Pten Δ/Δ : n = 51, Pten Δ/Δ Prkci Δ/Δ : n = 34, Pten Δ/Δ Rb1 Δ/Δ : n = 30, Pten Δ/Δ Rb1 Δ/Δ Prkci Δ/Δ : n = 26, PRKCI -OE: n = 42, Control WCM154: n = 41 cells examined). Scale bars, 10 μm. j , k Dose-response curves using CFU assay for 14 days to determine IC 50 of ENZA for sgC and sg PRKCI LNCaP cells transduced with the indicated siRNAs or treated with 10 μM GSK126. IC 50 value is the average of two biological replicates. Data shown as mean ± SD ( d , e ) and mean ± SEM ( g , h , i ) of 3 biological replicates. Two-tailed unpaired Student’s t-test ( d , e , g , h , i ). Source data are provided as a file.

Article Snippet: The following antibodies were used: CUTANA™ IgG Negative Control Antibody for CUT&RUN and CUT&Tag (#13-0042, 1:50), rabbit anti-H3K4me3 SNAP-Certified™ for CUT&RUN (#13-0041, 1:50), rabbit anti-EZH2 CUTANA™ CUT&RUN (#13-2026, 1:50), rabbit anti-H3K27me3 SNAP-Certified™ for CUT&RUN and CUT&Tag (#13-0055, 1:50) from Epicypher and rabbit anti-YY1 (#46395, 1:50) from Cell Signaling Technology.

Techniques: Generated, Western Blot, Extraction, Control, Colony-forming Unit Assay, Transduction, Two Tailed Test

Journal: Nature Communications

Article Title: Increased translation driven by non-canonical EZH2 creates a synthetic vulnerability in enzalutamide-resistant prostate cancer

doi: 10.1038/s41467-024-53874-2

Figure Lengend Snippet: a findGO.pl analysis of upregulated genes that exhibit EZH2 solo cobound with YY1 in sg PRKCI cells treated with 10 μM ENZA for 72 h (n = 3 biological replicates). b GSEA from RNA-seq of sg PRKCI and sgC LNCaP cells treated as in ( a ) using C2, and C5 gene sets (n = 3 biological replicates). FDR, false discovery rate. c EZH2, YY1, H3K4me3, and H3K27me3 binding (depth normalized) at the indicated loci in sg PRKCI and sgC LNCaP cells treated as in ( a ) (n = 3 biological replicates). d qPCR from sg PRKCI and sgC LNCaP cells, transduced with siRNAs, and treated as in ( a ) (n = 3 biological replicates). e Immunoblots in sg PRKCI and sgC LNCaP cells, transduced with siRNAs, and treated as in ( a ) (n = 2 independent experiments). f Dot plot pathway enrichment map showing the pathway enriched in each cluster of TRAMP + (n = 1 tumor sample from 1 mouse) and TRAMP + Prkci f/f PbCre + mice (n = 3 tumor samples from 1 mouse). g Puromycylation assay. Cells were stimulated with 1 μM Puromycin for 30 min, and puromycin-incorporated peptides were detected by immunoblotting. h Puromycylation assay in sg PRKCI and sgC LNCaP cells, treated as in ( a ), and quantification (n = 3 independent experiments). i Puromycin staining in sg PRKCI and sgC LNCaP cells, treated as in ( a ) (n = 3 biological replicates). Scale bars, 10 μm. j , k Puromycylation assay in Pten Δ/Δ and Pten Δ/ Δ Prkci Δ/Δ prostate organoids mouse ( j ) or EZH2 WT and EZH2 S375/380AA LNCaP cells ( k ) treated as in ( a ), and quantification (n = 3 independent experiments). l – n Puromycylation assay in sg PRKCI and sgC LNCaP cells, treated with 10 μM ENZA for 72 h, and 0,2, and 4 μM MS1943 ( l ), or transduced with the indicates siRNAs ( m ), or treated with 10 μM GSK126 ( n ), and quantification (n = 3 independent experiments). Data shown as mean ± SEM of 3 biological replicates ( d , h , i , j , k , l , m , n ). Two-tailed unpaired Student’s t-test ( d , h , i , j , k , l , m , n ). Source data are provided as a file.

Article Snippet: The following antibodies were used: CUTANA™ IgG Negative Control Antibody for CUT&RUN and CUT&Tag (#13-0042, 1:50), rabbit anti-H3K4me3 SNAP-Certified™ for CUT&RUN (#13-0041, 1:50), rabbit anti-EZH2 CUTANA™ CUT&RUN (#13-2026, 1:50), rabbit anti-H3K27me3 SNAP-Certified™ for CUT&RUN and CUT&Tag (#13-0055, 1:50) from Epicypher and rabbit anti-YY1 (#46395, 1:50) from Cell Signaling Technology.

Techniques: RNA Sequencing Assay, Binding Assay, Transduction, Western Blot, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: Increased translation driven by non-canonical EZH2 creates a synthetic vulnerability in enzalutamide-resistant prostate cancer

doi: 10.1038/s41467-024-53874-2

Figure Lengend Snippet: a Schematic of monosome and polysome isolation by sucrose gradient fractionation. b Polysome profiles of sg PRKCI and sgC LNCaP cells treated with 10 μM of ENZA for 72 h (n = 3 biological replicates). c Top 5 Hallmark pathways enriched in translationally efficient mRNAs upregulated in LNCaP sg PRKCI , by HOMER software (n = 3 biological replicates). d Ingenuity Pathway Analysis for translationally efficient mRNAs of LNCaP sg PRKCI vs sgC determined by Xtail (n = 3 biological replicates). e Volcano plot of translationally efficient mRNAs of LNCaP sg PRKCI vs sgC determined by Xtail (n = 3 biological replicates) (Blue= neuronal genes, red= EMT-related genes). f Immunoblots in sg PRKCI and sgC LNCaP cells transduced with the indicated siRNAs, treated as in ( a ) and quantification (n = 3 independent experiments). g Upstream regulator analysis of translationally efficient genes enriched mRNAs in sg PRKCI , treated with ENZA for 72 h (n = 3 biological replicates). h Dose-response curves to determine the IC 50 of ENZA either treated with vehicle or 20 μM galunisertib (Gal) in sgC and sg PRKCI LNCaP cells for 6 days. IC 50 value is the average of two biological replicates. i Growth curves of sgC and sg PRKCI LNCaP cells treated with 10 μM of ENZA and 20 μM of galunisertib alone or combined. Representative experiment of two biological replicates. j Growth curves of PRKCI -overexpressing ( PRKCI -OE) WCM1078 organoids treated as in ( i ). Representative experiment of three biological replicates. k PKCλ/ι’s dual role in EZH2 regulation. First, by controlling its stability, mediating its interaction with RBBP6. Second, by facilitating the transition of EZH2 from a Polycomb repressor to a transcriptional coactivator of YY1. This transition mediates resistance to enzalutamide induced by the loss of PKCλ/ι. Data shown as mean ± SEM of 3 biological replicates ( f ), mean ± SD of technical triplicates ( i) , mean ± SD of 3 biological replicates ( j ). Two-way ANOVA ( i , j ). Two-tailed unpaired Student’s t-test ( f ). Source data are provided as a file.

Article Snippet: The following antibodies were used: CUTANA™ IgG Negative Control Antibody for CUT&RUN and CUT&Tag (#13-0042, 1:50), rabbit anti-H3K4me3 SNAP-Certified™ for CUT&RUN (#13-0041, 1:50), rabbit anti-EZH2 CUTANA™ CUT&RUN (#13-2026, 1:50), rabbit anti-H3K27me3 SNAP-Certified™ for CUT&RUN and CUT&Tag (#13-0055, 1:50) from Epicypher and rabbit anti-YY1 (#46395, 1:50) from Cell Signaling Technology.

Techniques: Isolation, Fractionation, Software, Western Blot, Transduction, Two Tailed Test

Journal: Journal for immunotherapy of cancer

Article Title: Oncolytic peptide LTX-315 induces anti-pancreatic cancer immunity by targeting the ATP11B-PD-L1 axis.

doi: 10.1136/jitc-2021-004129

Figure Lengend Snippet: Figure 5 LTX-315-reduced ATP11B is a potential regulator of cancer immunity and PD-L1 expression. (A–B) Overall survival analysis in CD8 +T cell enriched (A) and CD8 +T cell decreased (B) patients with pancreatic cancer with low or high ATP11B expression. (C–E) Positive correlation of ATP11B with PD-L1 in pancreatic cancer. Representative images (C−D) and statistical results (E) of ATP11B and PD-L1 IHC staining in pancreatic cancer tissue microarray. (F–G) Correlation between the expression of ATP11B and abundance of immunoinhibitors (F) as well as tumor-infiltrating lymphocytes (G) across multiple human cancers, as analyzed on the The Cancer Genome Atlas database. (H–M) Maintenance of PD-L1 expression by ATP11B in multiple pancreatic cancer cell lines. Western blotting of PD-L1 expression in ATP11B KD/KO pancreatic cancer cell lines (H−I) and cell lines overexpressing ATP11B (J−K). Flow cytometry showing PD-L1 expression in ATP11B KD/KO pancreatic cancer cell lines and cell lines overexpressing ATP11B (L−M). IHC, immunohistochemical; KD, knockdown; KO, knockout; PD-L1, programmed cell death ligand 1; WT, wild type.

Article Snippet: MATERIALS AND METHODS Antibodies, inhibitors, and agents The following antibodies were used for immunoprecipitation/immunoblotting: rabbit anti- ATP11B (SAB3500528, Sigma- Aldrich), rabbit anti- ATP11B (13 672–1- AP, Proteintech), mouse anti-β actin (ab8226, Abcam), rabbit anti- CKLF- like MARVEL transmembrane domain containing 6 (CMTM6) (HPA026980, Sigma- Aldrich), rabbit anti- CMTM6 (55829, Cell Signaling Technology), rabbit anti- CMTM6 (DF3944, Affinity), mouse anti- PD- L1 (66 248–1- Ig, Proteintech), and rabbit anti- PD- L1 (13684, Cell Signaling Technology).

Techniques: Expressing, Immunohistochemistry, Microarray, Western Blot, Flow Cytometry, Immunohistochemical staining, Knockdown, Knock-Out

Journal: Journal for immunotherapy of cancer

Article Title: Oncolytic peptide LTX-315 induces anti-pancreatic cancer immunity by targeting the ATP11B-PD-L1 axis.

doi: 10.1136/jitc-2021-004129

Figure Lengend Snippet: Figure 6 ATP11B interacts with PD-L1 in a CMTM6-dependent manner. (A–B) ATP11B interaction with PD-L1 in vivo. Cell lysates from KPC and SW1990 cells were separately subjected to immunoprecipitation and western blotting using the indicated antibodies. (C) Lack of binding of ATP11B to PD-L1 in vitro. FLAG-tagged ATP11B and MYC-tagged PD-L1 purified from 293 T cell lysates and subjected to immunoprecipitation after co-incubation. (D) Positive correlation of ATP11B with CMTM6. Correlation of ATP11B and CMTM6 analyzed according to the pancreatic data sets of The Cancer Genome Atlas. (E–G) Representative images (E–F) and statistical results (G) of CMTM6 and ATP11B immunohistochemical staining in pancreatic cancer tissue microarray. (H) Western blotting of ATP11B, CMTM6, and PD-L1 expression in paired clinical tissue samples. (I–M) Interaction of ATP11B with PD-L1 in pancreatic cancer in a CMTM6-dependent manner. (I−J) cell lysates from KPC and SW1990 cells were separately subjected to immunoprecipitation and western blotting using the indicated antibodies. (K) Western blotting of ATP11B expression in pancreatic cancer cell lines after CMTM6 knockdown. (L–M) Cell lysates from CMTM6-KD KPC (L) and CMTM6-KD SW1990 (M) separately subjected to immunoprecipitation and western blotting using the indicated antibodies. CMTM6, CKLF-like MARVEL transmembrane domain containing 6; PD-L1 programmed cell death ligand 1.

Article Snippet: MATERIALS AND METHODS Antibodies, inhibitors, and agents The following antibodies were used for immunoprecipitation/immunoblotting: rabbit anti- ATP11B (SAB3500528, Sigma- Aldrich), rabbit anti- ATP11B (13 672–1- AP, Proteintech), mouse anti-β actin (ab8226, Abcam), rabbit anti- CKLF- like MARVEL transmembrane domain containing 6 (CMTM6) (HPA026980, Sigma- Aldrich), rabbit anti- CMTM6 (55829, Cell Signaling Technology), rabbit anti- CMTM6 (DF3944, Affinity), mouse anti- PD- L1 (66 248–1- Ig, Proteintech), and rabbit anti- PD- L1 (13684, Cell Signaling Technology).

Techniques: In Vivo, Immunoprecipitation, Western Blot, Binding Assay, In Vitro, Purification, Incubation, Immunohistochemical staining, Staining, Microarray, Expressing, Knockdown

Journal: Journal for immunotherapy of cancer

Article Title: Oncolytic peptide LTX-315 induces anti-pancreatic cancer immunity by targeting the ATP11B-PD-L1 axis.

doi: 10.1136/jitc-2021-004129

Figure Lengend Snippet: Figure 7 ATP11B prevents lysosomal degradation of PD-L1 through the interaction with CMTM6. (A–B) regulation of PD-L1 expression by ATP11B via lysosome-mediated degradation. (A) PD-L1 expression was analyzed by western blotting in WT and ATP11B KO KPC treated with or without the proteasome inhibitor MG132. (B) PD-L1 expression was analyzed by western blotting in WT and ATP11B KPC KO treated with or without the lysosome inhibitor aloxistatin and pepstatin A. (C–F) regulation of CTMT6 expression by ATP11B in pancreatic cancer. CMTM6 expression was analyzed by western blotting in pancreatic cancer cell lines overexpressing ATP11B (C–D) and ATP11B KD/KO pancreatic cell lines (E–F). (G–K) Rescues of the decrease of PD-L1 by CMTM6 overexpression caused by ATP11B kD. Western blotting (G–I) and flow cytometry (J–K) analysis of PD-L1 expression in WT/ATP11B kD pancreatic cancer cell lines with or without CMTM6 overexpression. CMTM6, CKLF-like MARVEL transmembrane domain containing 6; KD, knockdown; KO, knockout; PD-L1, programmed cell death ligand 1; WT, wild type.

Article Snippet: MATERIALS AND METHODS Antibodies, inhibitors, and agents The following antibodies were used for immunoprecipitation/immunoblotting: rabbit anti- ATP11B (SAB3500528, Sigma- Aldrich), rabbit anti- ATP11B (13 672–1- AP, Proteintech), mouse anti-β actin (ab8226, Abcam), rabbit anti- CKLF- like MARVEL transmembrane domain containing 6 (CMTM6) (HPA026980, Sigma- Aldrich), rabbit anti- CMTM6 (55829, Cell Signaling Technology), rabbit anti- CMTM6 (DF3944, Affinity), mouse anti- PD- L1 (66 248–1- Ig, Proteintech), and rabbit anti- PD- L1 (13684, Cell Signaling Technology).

Techniques: Expressing, Western Blot, Over Expression, Flow Cytometry, Knockdown, Knock-Out

Journal: Journal for immunotherapy of cancer

Article Title: Oncolytic peptide LTX-315 induces anti-pancreatic cancer immunity by targeting the ATP11B-PD-L1 axis.

doi: 10.1136/jitc-2021-004129

Figure Lengend Snippet: Figure 8 ATP11B depletion activates anti-pancreatic cancer immunity. (A–F) Inhibition of pancreatic tumor growth by ATP11B depletion in an immune system-dependent manner. (A–B) WT and ATP11B KO KPC cells separately orthotopically inoculated into the immunodeficient and immunocompetent mice. Survival curves of immunodeficient (A) and immunocompetent mice (B). WT and ATP11B KO KPC cells were separately orthotopically inoculated into the immunodeficient and immunocompetent mice (n=6). Representative images and weight of tumors in the immunodeficient (C) and immunocompetent mice (D) were individually recorded at the experimental endpoints. Representative FACS images of tumor-infiltrating lymphocytes in immunocompetent mice were shown and further quantified (E–F). (G) Representative images of PD-L1 immunohistochemical staining shown and further quantified. (H–L) Abolition of the therapeutic efficacy of LTX-315 in pancreatic cancer by ATP11B KO. WT and ATP11B KO KPC cells subcutaneous inoculated into the immunocompetent mice (n=5) treated with LTX-315. Growth curves of tumors were recorded at the indicated time points (H). Representative images of tumor weight (I) and tumors (J) were individually recorded at the experimental endpoints. Representative images of FACS analysis of T cell infiltration (K) and T cell function (L) were individually shown and further quantified. Results are presented as mean±SD from one representative experiment. *p<0.05, **p<0.01, ***p<0.001 by a two-tailed t-test; KO, knockout; NS, not significant; PD-L1, programmed cell death ligand 1; WT, wild type.

Article Snippet: MATERIALS AND METHODS Antibodies, inhibitors, and agents The following antibodies were used for immunoprecipitation/immunoblotting: rabbit anti- ATP11B (SAB3500528, Sigma- Aldrich), rabbit anti- ATP11B (13 672–1- AP, Proteintech), mouse anti-β actin (ab8226, Abcam), rabbit anti- CKLF- like MARVEL transmembrane domain containing 6 (CMTM6) (HPA026980, Sigma- Aldrich), rabbit anti- CMTM6 (55829, Cell Signaling Technology), rabbit anti- CMTM6 (DF3944, Affinity), mouse anti- PD- L1 (66 248–1- Ig, Proteintech), and rabbit anti- PD- L1 (13684, Cell Signaling Technology).

Techniques: Inhibition, Immunohistochemical staining, Staining, Drug discovery, Cell Function Assay, Two Tailed Test, Knock-Out

Journal: Nature Communications

Article Title: Functional screening implicates miR-371-3p and peroxiredoxin 6 in reversible tolerance to cancer drugs

doi: 10.1038/ncomms12351

Figure Lengend Snippet: ( a ) Downregulation of luciferase by miR-371-3p upon erlotinib treatment from 3′UTR reporters corresponding to candidate miR-371-3p target genes * P <0.05. ( b ) Microarray analysis of mRNA expression of miR-371-3p targets in pre-miR-371-expressing PC9 cells * P <0.05. ( c ) siRNA screen data corresponding to miR-371-3p targets plotted as z scores of siRNAs in erlotinib ( y axis) versus DMSO ( x axis)-treated cells. ( d ) Validation of siRNA knockdown effects of miR-371-3p targets, PRDX6 , PLCβ4 and STX12 on DTPs from PC9 cells. * and ** indicate significant differences from DMSO- and erlotinib-treated NTC controls, respectively. ( e ) Quantitative PCR of gene expression of PRDX6 , PLCβ4 and STX12 in pre-miR-371-expressing PC9 cells. ( f ) Mutations of seed sequences of the putative miR-371-3p recognition element in target 3′UTR's prevented inhibition by miR-371-3p. ( g ) PRDX6 , PLCβ4 and STX12 mRNA, and ( h ) protein expression in PC9 parental cells and DTPs. ( i ) DTP count of PC9 cells with PRDX6 , PLCβ4 and STX12 CRISPR/Cas9 knockouts, and ( j ) PC9 cells expressing sh PRDX6 , sh PLCβ4 and sh STX12 single, double or triple knockdowns. * indicates significant differences from wild-type (WT) controls. All experiments were performed in triplicate and data are representative of at least two independent experiments. Data are represented as mean±s.e.m. * and ** P <0.05, Student's t -test. NS, not significant.

Article Snippet: Immunoblotting was performed using 1:1,000 dilutions of the following antibodies, p-PKCα (sc-12356, Santa Cruz Biotechnology), PKCα (sc-208, Santa Cruz Biotechnology), PRDX6 (13585-1-AP, ProteinTech), PLCβ4 (sc-404, Santa Cruz Biotechnology), STX12 (Syntaxin 12) (sc-368438, Santa Cruz Biotechnology) and β-actin (3700S, Cell Signaling).

Techniques: Luciferase, Microarray, Expressing, Biomarker Discovery, Knockdown, Real-time Polymerase Chain Reaction, Gene Expression, Inhibition, CRISPR

Journal: Nature Communications

Article Title: Functional screening implicates miR-371-3p and peroxiredoxin 6 in reversible tolerance to cancer drugs

doi: 10.1038/ncomms12351

Figure Lengend Snippet: ( a ) Co-treatment with bisindolylmaleimide XI reduces DTPs in PC9, COLO-205 and HCC-1954 cells. ( b ) Co-treatment with cinnamycin reduces DTPs in COLO-205, HCC-1954 and MKN-45 cells. Decreased PLA2 activity upon ( c ) siRNA knockdown of PRDX6 , PLCβ4 or STX12 and ( d ) treatment with PLA2 inhibitors cinnamycin and the PKCα inhibitor bisindolylmaleimide XI. * indicates significant differences from wild-type (WT) or DMSO controls. ( e ) PKC activity in PC9 cells and DTPs, stable cell lines expressing pre-miR-371 or PRDX6 , PLCβ4 or STX12 knockouts and upon treatment with bisindolylmaleimide XI±erlotinib. * indicates significant differences from DMSO or WT controls. ( f ) Immunoblot of PKCα activation in PC9 DTPs and PC9 cells with PRDX6 , PLCβ4 or STX12 knockouts. ( g ) si PRKCA knockdown in PC9 cells reduces DTPs. * and ** indicate significant differences from DMSO- and erlotinib-treated NTC controls respectively. ( h ) Co-treatment with the PKC activator phorbol-12-myristate13-acetate (PMA) increases DTPs in PC9 cells i , Fluorescence microscopy images of surviving PC9 DTPs co-cultured with parental cells (T: top panel), PC9 DTPs (M: middle panel) or parental cells (B: bottom panel) after 6-day treatment with cinnamycin±erlotinib, Scale bar, 50 μm and ( j ) viable cell population counts of imaged cells in f on day 6 of treatment. All experiments were performed in triplicate and data are representative of at least two independent experiments. Data are represented as mean±s.e.m. For c – e and g , * or ** P <0.05, Student's t -test, NS, not significant. For a , b and h , P represents interaction P value.

Article Snippet: Immunoblotting was performed using 1:1,000 dilutions of the following antibodies, p-PKCα (sc-12356, Santa Cruz Biotechnology), PKCα (sc-208, Santa Cruz Biotechnology), PRDX6 (13585-1-AP, ProteinTech), PLCβ4 (sc-404, Santa Cruz Biotechnology), STX12 (Syntaxin 12) (sc-368438, Santa Cruz Biotechnology) and β-actin (3700S, Cell Signaling).

Techniques: Activity Assay, Knockdown, Stable Transfection, Expressing, Western Blot, Activation Assay, Fluorescence, Microscopy, Cell Culture

Journal: Nature Communications

Article Title: Functional screening implicates miR-371-3p and peroxiredoxin 6 in reversible tolerance to cancer drugs

doi: 10.1038/ncomms12351

Figure Lengend Snippet: ( a ) DTEP formation after erlotinib treatment is reduced in PC9 cells on pre-miR-371 overexpression, single, double or triple knockdowns of PRDX6 , PLCβ4 or STX12 , knockouts of PRDX6 , PLCβ4 or STX12 and treatment with PLA2/PKCα inhibitors or phorbol-12-myristate13-acetate (PMA; n =3). ( b ) DTEP colony counts obtained in a . Data are representative of at least two independent experiments. Data are represented as mean±s.e.m. * ( P <0.05, Student's t -test) indicate significant differences from wild-type (WT) controls. ( c ) Xenograft studies with PC9+GFP and PC9+pre-miR-371 cell lines using erlotinib ( n =15). Data are represented as mean±s.e.m. Interaction P values * P =0.0288, ** P =5.17e−05, *** P =0.00408. ( d ) Change in tumour volumes during drug treatment and after treatment cessation. ( e ) Quantitative PCR for expression of PRDX6 , PLCβ4 and STX12 mRNAs in xenograft tumours ( n =5). * P <0.05, Student's t -test. ( f ) Kaplan–Meier plots of overall survival of lung adenocarcinoma, colorectal cancer, gastric cancer and breast cancer patients; patient groups were separated based on PRDX6 mRNA expression. ( g ) Model depicting proposed mechanism for regulation of DTP survival via miR-371-3p and PRDX6 .

Article Snippet: Immunoblotting was performed using 1:1,000 dilutions of the following antibodies, p-PKCα (sc-12356, Santa Cruz Biotechnology), PKCα (sc-208, Santa Cruz Biotechnology), PRDX6 (13585-1-AP, ProteinTech), PLCβ4 (sc-404, Santa Cruz Biotechnology), STX12 (Syntaxin 12) (sc-368438, Santa Cruz Biotechnology) and β-actin (3700S, Cell Signaling).

Techniques: Over Expression, Real-time Polymerase Chain Reaction, Expressing

Journal: Frontiers in Immunology

Article Title: Hspa13 Promotes Plasma Cell Production and Antibody Secretion

doi: 10.3389/fimmu.2020.00913

Figure Lengend Snippet: Hspa13 mRNA was increased in atacicept-induced plasmablasts (PBs) and plasma cells (PCs).

Article Snippet: The blots were then incubated overnight at 4°C with rabbit antibodies against Hspa13 (Cat no. 12667-2-AP, Proteintech Group Inc.) and β-tubulin (KM9003T, SunGene Biotech) antibodies diluted 1:1,000 in TBS-T containing 5% bovine serum albumin.

Techniques: Clinical Proteomics, Marker

Journal: Frontiers in Immunology

Article Title: Hspa13 Promotes Plasma Cell Production and Antibody Secretion

doi: 10.3389/fimmu.2020.00913

Figure Lengend Snippet: Hspa13 mRNA was increased in LPS-induced PBs/PCs.

Article Snippet: The blots were then incubated overnight at 4°C with rabbit antibodies against Hspa13 (Cat no. 12667-2-AP, Proteintech Group Inc.) and β-tubulin (KM9003T, SunGene Biotech) antibodies diluted 1:1,000 in TBS-T containing 5% bovine serum albumin.

Techniques: Microarray, Marker

Journal: Frontiers in Immunology

Article Title: Hspa13 Promotes Plasma Cell Production and Antibody Secretion

doi: 10.3389/fimmu.2020.00913

Figure Lengend Snippet: Hspa13 was highly expressed in plasmablasts (PBs) and plasma cells (PCs). (A–C) Hspa13 expression was up-regulated in LPS-induced PBs/PCs. The splenocytes were separated from six 7- to 9-week-old C57BL/6 mice. B cells were sorted by B220 microbeads and stimulated for 0, 24, 48, and 72 hours (hrs) in vitro by 10 μg/ml LPS. Cells were collected and subjected to qPCR (A) , RT-PCR/agarose (B) , and western blot (C) analysis. DNA band ( B , upper panel) was verified as Hspa13 by DNA sequencing. (D,E) Hspa13 was highly expressed in PBs and PCs but not in naïve B cells or germinal center (GC) B cells. Lymphocytes were collected from splenocytes and lymph nodes (LNs) of six sheep red cell (SRC)-immunizated C57BL/6 mice described in . Naïve B cells (CD19 + B220 + IgM + IgD + ), GC B cells (CD19 + B220 + GL7 + CD38 low ), PBs (TACI + CD138 + B220 int CD19 int ), and PCs (TACI + CD138 + B220 − CD19 − ) were sorted by flow cytometry (FACS) and described in , and subjected to qPCR (D) and western blot (E) analysis. (A–E) Data represent three independent experiments with six individual mice each. (A,D) Data were analyzed by the one-way ANOVA plus the Bonferroni test: compare selected pairs of columns and show as mean ± s.e.m ( N = 6 for all groups). ** P < 0.01, *** P < 0.001.

Article Snippet: The blots were then incubated overnight at 4°C with rabbit antibodies against Hspa13 (Cat no. 12667-2-AP, Proteintech Group Inc.) and β-tubulin (KM9003T, SunGene Biotech) antibodies diluted 1:1,000 in TBS-T containing 5% bovine serum albumin.

Techniques: Clinical Proteomics, Expressing, In Vitro, Reverse Transcription Polymerase Chain Reaction, Western Blot, DNA Sequencing, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Hspa13 Promotes Plasma Cell Production and Antibody Secretion

doi: 10.3389/fimmu.2020.00913

Figure Lengend Snippet: Hspa13 mRNA was expressed in PBs.

Article Snippet: The blots were then incubated overnight at 4°C with rabbit antibodies against Hspa13 (Cat no. 12667-2-AP, Proteintech Group Inc.) and β-tubulin (KM9003T, SunGene Biotech) antibodies diluted 1:1,000 in TBS-T containing 5% bovine serum albumin.

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Hspa13 Promotes Plasma Cell Production and Antibody Secretion

doi: 10.3389/fimmu.2020.00913

Figure Lengend Snippet: CD19 cre Hspa13 fl/fl mice were developed. (A) A construction map of Hspa13 fl/fl and CD19 cre Hspa13 fl/fl mice. This project used the principle of homologous recombination and adopted embryonic stem (ES) cell targeting to modify the Hspa13 locus (Chr16:75755190-75767276 bp) by flox modification. The brief process is as follows: The BAC clone containing the gene of interest was purchased from the Sanger Institute (UK). The ES cell targeting vector was constructed by the ET-clone method. The vector contains a 3.4 Kb 5′ homology arm, a 541 bp flox region, and a PGK-neo-polyA, 3.6 kb 3′ homology arm, plus MC1-TK-polyA negative selection marker. After the vector was linearized, JM8A3 ES cells were transfected electrically. A total of 96 resistant clones were obtained after screening with the G418 and Ganc drugs. A total of 13 positive clones with correct homologous recombination were identified by long fragment PCR. Positive ES cell clones were expanded and injected into blastocysts of C57BL/6J mice to obtain chimeric mice. A high proportion of chimeric mice were mated with C57BL/6J mice to obtain seven positive F1 mice. Hspa13 gene flox heterozygous mice showed no significant abnormalities. After mating the flox mouse with a heterologous CD19 cre mouse, the progeny of the flox homozygous, Cre-positive mouse was knocked out, resulting in a functional loss of the gene of interest in B cells. (B) Hspa13 was knocked out in PBs/PCs from CD19 cre Hspa13 fl/fl mice. PCs (TACI + CD138 + B220 − CD19 − ) were sorted from the spleens and bone marrows (BMs) of 7- to 9-week-old heterologous CD19 cre , Hspa13 fl/fl , and CD19 cre Hspa13 fl/fl mice by FACS. PCs were subjected to PCR (B) and western blot (C) analysis. PCR products: with cre activity: 1,468 bp; with no cre activity: 2,120 bp; wild type: 1,897 bp. (B,C) Data represent three independent experiments with three individual mice each.

Article Snippet: The blots were then incubated overnight at 4°C with rabbit antibodies against Hspa13 (Cat no. 12667-2-AP, Proteintech Group Inc.) and β-tubulin (KM9003T, SunGene Biotech) antibodies diluted 1:1,000 in TBS-T containing 5% bovine serum albumin.

Techniques: Homologous Recombination, Modification, Plasmid Preparation, Construct, Selection, Marker, Transfection, Clone Assay, Injection, Functional Assay, Western Blot, Activity Assay

Journal: Frontiers in Immunology

Article Title: Hspa13 Promotes Plasma Cell Production and Antibody Secretion

doi: 10.3389/fimmu.2020.00913

Figure Lengend Snippet: PBs, PCs, and antibodies were reduced in CD19 cre Hspa13 fl/fl (Hspa13 cKO) mice. (A) Hspa13 cKO did not affect naïve B cells or germinal center (GC) B cells in mice. Splenic lymphocytes from 9-week-old wild type, Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice were separated using a lymphocyte separation solution; stained with isotype control antibodies, anti-mouse CD19, B220, CD38, and GL7 antibodies; and then analyzed by FACS. The percentages (left panel) and the absolute numbers (right panel) of CD19 + B220 + B cells and CD38 lo GL7 hi B220 + CD19 + GC B cells are shown. (B) Hspa13 cKO reduced PBs, early PCs, and mature PCs in mice. Lymphocytes from the spleen, LNs, and BMs of 9-week-old WT, Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice were separated using a lymphocyte separation solution; stained with isotype control antibodies, anti-mouse TACI, CD19, B220, and CD138 antibodies; and then analyzed by FACS. The percentages (left panel) and the absolute numbers (right panel) of TACI + CD138 + B220 int CD19 int PBs, TACI + CD138 + B220 − CD19 int early PCs, and TACI + CD138 + B220 − CD19 − mature PCs are shown. (C) Hspa13 cKO reduced antibodies in mice. Sera were collected from 9-week-old WT, Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice, and the total IgM, IgG, IgG1, IgG2b, IgG2c, IgG3, IgA, and IgE antibody levels were analyzed by ELISA. (A–C) Data represent three independent experiments, with six mice per group per experiment. Data were analyzed by the one-way ANOVA plus the Bonferroni test: compare selected pairs of columns and show as mean ± s.e.m ( N = 6 for all groups). * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The blots were then incubated overnight at 4°C with rabbit antibodies against Hspa13 (Cat no. 12667-2-AP, Proteintech Group Inc.) and β-tubulin (KM9003T, SunGene Biotech) antibodies diluted 1:1,000 in TBS-T containing 5% bovine serum albumin.

Techniques: Staining, Control, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Hspa13 Promotes Plasma Cell Production and Antibody Secretion

doi: 10.3389/fimmu.2020.00913

Figure Lengend Snippet: Hspa13 cKO reduced the production of LPS-induced PBs, PCs, and antibodies. Splenic B cells from 9-week-old Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice were sorted with B220 microbeads and were then stimulated with 10 μg/ml LPS for 0, 1, 2, and 3 days. (A) Hspa13 cKO did not affect LPS-stimulated B-cell proliferation. On days 0, 1, 2, and 3 following LPS stimulation, a CCK8 assay was used to evaluate the cell proliferation. (B,C) Hspa13 cKO did not affect LPS-stimulated B-cell activation. On day 3 following LPS stimulation, cells were stained with isotype control antibodies, anti-mouse B220 and GL7 antibodies, and analyzed by FACS. The percentages (B) and the absolute numbers (C) of B220 + GL7 + B cells are shown. (D,E) Hspa13 cKO reduced LPS-induced PBs, early PCs, and mature PCs. On day 3 following LPS stimulation, cells were stained with isotype control antibodies, anti-mouse TACI, CD19, B220, and CD138 antibodies, and were then analyzed by FACS. The percentages (D) and the absolute numbers (E) of TACI + CD138 + B220 int CD19 int PBs, TACI + CD138 + B220 − CD19 int early PCs, and TACI + CD138 + B220 − CD19 − mature PCs are shown. (F) Hspa13 cKO reduced LPS-induced antibody secretion. On day 3 following LPS stimulation, culture supernatants were collected and the total IgM, IgG1, IgG2b, IgG2c, and IgG3 antibody levels were analyzed by ELISA. (A–F) Data represent three independent experiments, with six mice per group per experiment. Data were analyzed by two-way (A) and one-way (C,E,F) ANOVA plus the Bonferroni test: compare selected pairs of columns and show as mean ± s.e.m ( N = 6 for all groups). *** P < 0.001.

Article Snippet: The blots were then incubated overnight at 4°C with rabbit antibodies against Hspa13 (Cat no. 12667-2-AP, Proteintech Group Inc.) and β-tubulin (KM9003T, SunGene Biotech) antibodies diluted 1:1,000 in TBS-T containing 5% bovine serum albumin.

Techniques: CCK-8 Assay, Activation Assay, Staining, Control, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Hspa13 Promotes Plasma Cell Production and Antibody Secretion

doi: 10.3389/fimmu.2020.00913

Figure Lengend Snippet: Hspa13 deficiency reduced Prdm1 and Xbp1 expression.

Article Snippet: The blots were then incubated overnight at 4°C with rabbit antibodies against Hspa13 (Cat no. 12667-2-AP, Proteintech Group Inc.) and β-tubulin (KM9003T, SunGene Biotech) antibodies diluted 1:1,000 in TBS-T containing 5% bovine serum albumin.

Techniques: Expressing, Marker

Journal: Frontiers in Immunology

Article Title: Hspa13 Promotes Plasma Cell Production and Antibody Secretion

doi: 10.3389/fimmu.2020.00913

Figure Lengend Snippet: Hspa13 cKO reduced sheep red blood cell (SRC)-induced PB/PC and antibody production. Nine-week-old female Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice were injected intraperitoneally (i.p.) with 1 × 10 9 SRCs on days 0 and 7. (A,B) Hspa13 cKO did not affect SRC-induce GC B-cell production. On day 21 following SRC stimulation, splenic lymphocytes were stained with isotype control antibodies, anti-mouse CD19, B220, CD38, and GL7 antibodies, and were then analyzed by FACS. The percentages (A) and the absolute numbers (B) of CD38 lo GL7 hi GC cells gated on CD19 + B220 + are shown. (C,D) Hspa13 cKO did not affect the SRC-induced dark zone (DZ) and light zone (LZ) GC B-cell production. On day 21 following SRC stimulation, splenic lymphocytes were stained with anti-mouse CD19, B220, CD38, GL7, CXCR4, and CD86 antibodies, and were then analyzed by FACS. The percentages (C) and the absolute numbers (D) of CXCR4 hi CD86 lo DZ and CXCR4 lo CD86 hi LZ GC B cells gated on CD19 + B220 + CD38 lo GL7 hi GC cells are shown. (E,F) Hspa13 cKO reduced SRC-induced IgG1-, IgG2b-, IgG2c-, and IgG3-expressing PBs/PCs. On day 21 following SRC stimulation, splenic lymphocytes were collected and intracellular staining was performed with isotype control antibodies, anti-mouse B220, IgG1, IgG2b, IgG2c, and IgG3 antibodies. The percentages (E) and the absolute numbers (F) of IgG1-, IgG2b-, IgG2c-, and IgG3-expressing B220 + PBs and B220 − PCs are shown. (G) Hspa13 cKO reduced SRC-induced antibody secretion. On day 21 following SRC stimulation, sera were collected and the total IgM, IgG, IgG1, IgG2b, IgG2c, and IgG3 antibody levels were analyzed by ELISA. (A–G) Data represent three independent experiments, with six mice per group per experiment. Data were analyzed by two-way (D) and one-way (B,F,G) ANOVA plus the Bonferroni test: compare selected pairs of columns and show as mean ± s.e.m ( N = 6 for all groups). ** P < 0.01, *** P < 0.001.

Article Snippet: The blots were then incubated overnight at 4°C with rabbit antibodies against Hspa13 (Cat no. 12667-2-AP, Proteintech Group Inc.) and β-tubulin (KM9003T, SunGene Biotech) antibodies diluted 1:1,000 in TBS-T containing 5% bovine serum albumin.

Techniques: Injection, Staining, Control, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Hspa13 Promotes Plasma Cell Production and Antibody Secretion

doi: 10.3389/fimmu.2020.00913

Figure Lengend Snippet: Hspa13 cKO reduced 4-hydroxy-3-nitrophenylacetyl (NP)-specific antibody production. Nine-week-old Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice were injected i.p. with T cell-independent antigen NP-Ficoll (A) and T cell-dependent antigen NP-keyhole lymphocyte hemocyanin (KLH) (B) on days 0 and 7. On day 21 following NP-Ficoll (A) or NP-KLH (B) stimulation, sera were collected and the NP-specific IgM, IgG, IgG1, IgG2b, IgG2c, IgG3, IgA, and IgE antibody levels were analyzed by ELISA. (A,B) Data represent three independent experiments, with six mice per group per experiment. Data were analyzed by one-way ANOVA plus the Bonferroni test: compare selected pairs of columns and show as mean ± s.e.m ( N = 6 for all groups). * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The blots were then incubated overnight at 4°C with rabbit antibodies against Hspa13 (Cat no. 12667-2-AP, Proteintech Group Inc.) and β-tubulin (KM9003T, SunGene Biotech) antibodies diluted 1:1,000 in TBS-T containing 5% bovine serum albumin.

Techniques: Injection, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Hspa13 Promotes Plasma Cell Production and Antibody Secretion

doi: 10.3389/fimmu.2020.00913

Figure Lengend Snippet: Hspa13 cKO reduced class switch recombination (CSR), somatic hypermutation (SHM), and affinity maturation of antibodies. Nine-week-old female Hspa13 fl/fl (control) and CD19 cre Hspa13 fl/fl (Hspa13 cKO) mice (three mice per group) were injected i.p. with 1 × 10 9 SRCs (A–C) or NP-KLH (D,E) on days 0 and 7. On day 21 following SRC stimulation, splenocytes were stained with PerCP-conjugated anti-mouse B220 antibodies and sorted by FACS. Single cells were captured using the 10 X Genomics Full Chromium platform and subjected to RNA- and VDJ-sequencing. (A) Hspa13 cKO reduced SRC-induced PBs. Of the single PBs, 27 (3.49%) and 11 (1.07%) (Ighm + , Ighg1 + , Ighg2b + , Ighg2c + , Ighg3 + , Igha + , or Ighe + Cd3d − Cd3e − Cd3gCd4 − Cd8a − Cd19 + Ptprc + Ms4a1 + Ighd − Bcl6 − Aicda − Prdm1 + Xbp1 + Sdc1 + ) within the splenic B cell population were identified by single-cell RNA-sequencing out of 774 and 1,025 single cells corresponding to CD19 cre Hspa13 fl/fl and Hspa13 fl/fl mice, respectively. (B) Hspa13 cKO reduced SRC-induced antibody CSR. Single cells expressing genes encoding IgD, IgM, IgG1, IgG2b, IgG2c, IgG3, IgA, and IgE antibodies were identified by single-cell VDJ-sequencing. The percentage of different antibody subtypes expressed by single cells out of 734 and 382 antibody-expressing single cells from CD19 cre Hspa13 fl/fl and Hspa13 fl/fl mice, respectively, is shown. (C) Hspa13 cKO reduced SRC-induced antibody SHM. The single antibody gene was determined by single-cell VDJ-sequencing. SHM percentages in the CDR (complementarity-determining region) of the heavy (H) and light (L) chains are based on 382 and 734 antibody genes from Hspa13 fl/fl and CD19 cre Hspa13 fl/fl mice, respectively. (D) Hspa13 cKO reduced NP-specific SHM induced by NP-KLH. The distribution of the number of mutations per unique clone (VH186.2 segment) is shown. Numbers refer to 100 individual sequences; three animals per group were analyzed. (E) Hspa13 cKO reduced NP-specific high-affinity clones induced by NP-KLH. On day 21 following NP-KLH stimulation, the percentage of NP high-affinity clones containing the W33L mutation in CDR1 in purified GC B cells of Hspa13 fl/fl and CD19 cre Hspa13 fl/fl mice was determined. Each dot corresponds to a single animal (30 unique clones/mouse; Mann-Whitney test; error bars represent s.e.m; *** P < 0.001).

Article Snippet: The blots were then incubated overnight at 4°C with rabbit antibodies against Hspa13 (Cat no. 12667-2-AP, Proteintech Group Inc.) and β-tubulin (KM9003T, SunGene Biotech) antibodies diluted 1:1,000 in TBS-T containing 5% bovine serum albumin.

Techniques: Control, Injection, Staining, Sequencing, RNA Sequencing, Expressing, Clone Assay, Mutagenesis, Purification, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Hspa13 Promotes Plasma Cell Production and Antibody Secretion

doi: 10.3389/fimmu.2020.00913

Figure Lengend Snippet: Hspa13 interacts with endoplasmic reticulum (ER) proteins involved in positive regulation of protein transport from the ER to the cytosol. Splenic B220 + B cells from 9-week-old C57BL/6 mice (three mice per group) were sorted using B220 microbeads and were then stimulated for 3 days with 10 μg/ml LPS. LPS-stimulated B cells were collected for anti-Hspa13 antibody co-immunoprecipitation (IP) experiments. Co-immunoprecipitated proteins were identified by mass spectrometry. (A) SDS-PAGE and silver staining results showing affinity captured interacting proteins from whole cell extracts. Putative interacting protein bands marked as 1, 2, 3, and 4 were excised for mass spectrometry analysis. (B) Anti-Hspa13 antibody specifically co-immunoprecipitated 56 proteins. We identified 393, 56, and 148 proteins that were co-immunoprecipitated by control IgG antibodies only, anti-Hspa13 antibodies only, and the two antibodies together, respectively. (C) Bar plot ranking of the top 10 cellular components (CC), based on enrichment score. Gene ontology (GO)-analysis was performed using a gene ontology website ( http://www.geneontology.org/ ). (D) Bar plot ranking of the top 10 biologic processes (BP) based on enrichment score. GO-analysis was performed based on the gene ontology website ( http://www.geneontology.org/ ). (E) A list of the 10 best hits of Hspa13 interacting partners. Detailed interacting protein data are shown in . (F) Interaction of Hspa13 and Bcap31. The recombinant plasmids expressing Hspa13-V5 and Bcap31-Flag were transiently transfected into 293T cells. At 48 hrs after transfection, cells were lysed and anti-V5 antibody was used to immunoprecipitate proteins probed with anti-Flag antibody. Data are shown for one representative experiment from three independent experiments with similar results. (G) Hspa13 cKO reduced Bcap31 mRNA expression in PBs induced by SRC. Bcap31 mRNA expression was analyzed from 10 and 8 PBs from the single-cell RNA sequencing data of SRC-primed Hspa13 fl/fl and CD19 cre Hspa13 fl/fl mice, respectively, described in . Student's t -test (two tailed). Error bars represent s.e.m. * P < 0.05.

Article Snippet: The blots were then incubated overnight at 4°C with rabbit antibodies against Hspa13 (Cat no. 12667-2-AP, Proteintech Group Inc.) and β-tubulin (KM9003T, SunGene Biotech) antibodies diluted 1:1,000 in TBS-T containing 5% bovine serum albumin.

Techniques: Immunoprecipitation, Mass Spectrometry, SDS Page, Silver Staining, Control, Recombinant, Expressing, Transfection, RNA Sequencing, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Hspa13 Promotes Plasma Cell Production and Antibody Secretion

doi: 10.3389/fimmu.2020.00913

Figure Lengend Snippet: Hspa13 mRNA was increased in B220 + cells from patients with multiple myeloma (MM).

Article Snippet: The blots were then incubated overnight at 4°C with rabbit antibodies against Hspa13 (Cat no. 12667-2-AP, Proteintech Group Inc.) and β-tubulin (KM9003T, SunGene Biotech) antibodies diluted 1:1,000 in TBS-T containing 5% bovine serum albumin.

Techniques: Marker

Journal: Frontiers in Immunology

Article Title: Hspa13 Promotes Plasma Cell Production and Antibody Secretion

doi: 10.3389/fimmu.2020.00913

Figure Lengend Snippet: Hspa13 mRNA was increased in B220 + cells from patients with systemic lupus erythematosus (SLE).

Article Snippet: The blots were then incubated overnight at 4°C with rabbit antibodies against Hspa13 (Cat no. 12667-2-AP, Proteintech Group Inc.) and β-tubulin (KM9003T, SunGene Biotech) antibodies diluted 1:1,000 in TBS-T containing 5% bovine serum albumin.

Techniques: Marker

Journal: Frontiers in Immunology

Article Title: Hspa13 Promotes Plasma Cell Production and Antibody Secretion

doi: 10.3389/fimmu.2020.00913

Figure Lengend Snippet: Hspa13 cKO reduced autoantibodies and proteinuria in pristane-induced lupus and lupus-prone MRL/lpr mouse model. (A) Hspa13 cKO reduced autoantibodies in pristane-induced lupus mice. To induce lupus, 9-week-old female Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice on a B6 background were injected i.p. with 0.5 ml of pristane. On day 21 following pristane stimulation, sera were collected and dsDNA-specific IgM and IgG antibody levels were analyzed by ELISA. (B) Hspa13 cKO reduced proteinuria in the pristane-induced lupus mice. On day 21 following pristane stimulation, urine was collected and proteinuria was measured. (C) Hspa13 cKO reduced autoantibodies in a lupus-prone MRL/lpr mouse model. Sera were collected from Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice in lupus-prone MRL/lpr mice background at 6 months of age and dsDNA-specific IgM and IgG antibody levels were analyzed by ELISA. (D) Hspa13 cKO reduced proteinuria in the lupus-prone MRL/lpr mouse model. Urine was collected from Hspa13 fl/fl , CD19 cre , and CD19 cre Hspa13 fl/fl mice in a lupus-prone MRL/lpr mice background at 6 months of age and proteinuria was measured. (A–D) Data represent three independent experiments, with six mice per group per experiment. Data were analyzed by the one-way ANOVA plus the Bonferroni test: compare selected pairs of columns and show as mean ± s.e.m ( N = 6 for all groups). ** P < 0.01, *** P < 0.01.

Article Snippet: The blots were then incubated overnight at 4°C with rabbit antibodies against Hspa13 (Cat no. 12667-2-AP, Proteintech Group Inc.) and β-tubulin (KM9003T, SunGene Biotech) antibodies diluted 1:1,000 in TBS-T containing 5% bovine serum albumin.

Techniques: Injection, Enzyme-linked Immunosorbent Assay