Journal: Acta Crystallographica. Section F, Structural Biology Communications

Article Title: Neutron and high-resolution room-temperature X-ray data collection from crystallized lytic polysaccharide monooxygenase

doi: 10.1107/S2053230X15019743

Figure Lengend Snippet: Macromolecule-production information

Article Snippet: Details of the cloning and protein-production procedures are summarized in Table 1 . table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 Source organism J. denitrificans strain ATCC 14870 DNA source Synthetic DNA Forward primer 5-CCGTAGCAAT GGATCC ATGAAGAAGAGAAAGTTGAGAGCGTCAGC-3 Reverse primer 5-TCGTAATGCC GCGGCCGC TCATGAGACCACAACATCCATACAGTTG-3 Expression vector pUCBB-eGFP Expression host E. coli BL21 Star (DE3) Complete amino-acid sequence of the construct produced HGWVTDPPSRQALCASGETSFDCGQISYEPQSVEAPKGATTCSGGNEAFAILDDNSKPWPTTEIASTVDLTWKLTAPHNTSTWEYFVDGQLHQTFDQKGQQPPTSLTHTLTDLPTGEHTILARWNVSNTNNAFYNCMDVVVS Open in a separate window caption a8 Macromolecule-production information

Techniques: Expressing, Plasmid Preparation, Sequencing, Construct, Produced

Journal: eLife

Article Title: The domesticated transposon protein L1TD1 associates with its ancestor L1 ORF1p to promote LINE-1 retrotransposition

doi: 10.7554/elife.96850

Figure Lengend Snippet: Figure 1. DNA hypomethylation results in the activation of L1TD1 expression and loss of L1TD1 affects cell viability in HAP1 cells. (A) Quantification of DNA methylation levels at the L1TD1 promoter in HAP1 wildtype (WT), DNMT1 KO, and DNMT1/L1TD1 DKO cells using the MethyLight assay. DNA methylation is shown as percentage of methylation ratio (PMR). (B) qRT-PCR analysis of L1TD1 mRNA expression in HAP1 WT, DNMT1 KO, and DNMT1/ L1TD1 DKO cells. GAPDH was used as a normalization control and relative L1TD1 mRNA levels in DNMT1 KO cells were set to 1. Data are shown as a

Article Snippet: DOI: https://doi.org/10.7554/eLife.96850 13 of 22 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody DNMT1 (H- 300) (polyclonal rabbit) Santa Cruz sc20701 RRID:AB_2293064 1:1000 Antibody ɣ-H2AX (monoclonal mouse) Millipore JBW301 RRID:AB_2847865 1:1000 Antibody Histone H3 C- term (polyclonal rabbit) Abcam Ab1791 RRID:AB_302613 1:5000 Antibody Cleaved caspase 3 (polyclonal rabbit) Cell Signaling 9661 RRID:AB_2341188 1:50 Antibody Anti- mouse HRP (polyclonal) Jackson Laboratories 115-035-008 RRID:AB_2313585 1:10,000 Antibody Anti- rabbit HRP (polyclonal) Jackson Laboratories 211- 032- 171 RRID:AB_2339149 1:10,000 Antibody Goat anti- Rabbit IgG (H+L) Alexa Fluor Plus 488 Invitrogen A32731 RRID:AB_2633280 1:500 Antibody Goat anti- Mouse IgG (H+L) Alexa Fluor Plus 546 Invitrogen A11030 RRID:AB_2737024 1:500 Sequence- based reagent hL1TD1 _f This paper PCR primers CTTA CCCT GG TAGC CGAC CT Sequence- based reagent hL1TD1 _r This paper PCR primers GGCT GGCA AA TTTT CTAA GG Sequence- based reagent hARMC1_f This paper PCR primers AGCT CTGG AG CGAA TTTA AGA Sequence- based reagent hARMC1_r This paper PCR primers GGCA GACA TC CCTG ATCC TG Sequence- based reagent hYY2_f This paper PCR primers TCCC GGAT AG CATT GAAG AC Sequence- based reagent hYY2_r This paper PCR primers TTGA CCTG CA TTTG CTTC TG Sequence- based reagent hORF1p_f This paper PCR primers AGTG CTTA AAG GAGC TGAT GG Sequence- based reagent hORF1p_r This paper PCR primers AACT GGAA GAA AGGG TATC AGC Commercial assay or kit CellTiter- Glo Luminiscent Cell Viability Assay Promega G7571 Commercial assay or kit Monarch RNA Cleanup Kit New England Biolabs T2047L Commercial assay or kit Qubit RNA High Sensitivity kit Thermo Fisher Scientific Q32852 Commercial assay or kit iScript cDNA synthesis Kit Bio- Rad 1708891 Commercial assay or kit Wizard Genomic DNA isolation kit Promega Commercial assay or kit EZ DNA Methylation Kit Zymo Research D5001 Software, algorithm DESeq2 Love et al., 2014 RRID:SCR_015687 Software, algorithm TEtranscript Jin et al., 2015 RRID:SCR_023208 Continued on next page Continued Kavaklioglu et al. eLife 2024;13:RP96850.

Techniques: Activation Assay, Expressing, DNA Methylation Assay, Methylation, Quantitative RT-PCR, Control

Journal: eLife

Article Title: The domesticated transposon protein L1TD1 associates with its ancestor L1 ORF1p to promote LINE-1 retrotransposition

doi: 10.7554/elife.96850

Figure Lengend Snippet: Figure 3. L1TD1 cross-talk with its ancestor L1 ORF1p. (A) Volcano plot displaying the comparison of the proteomes of HAP1 DNMT1 KO and DNMT1/ L1TD1 DKO cells determined by mass spectrometry. Differentially abundant proteins were plotted as DNMT1/L1TD1 DKO over DNMT1 KO (log2FC ≥1, adj. p-value<0.05 [red] and log2FC ≤ –1, adj. p-value<0.05 [blue]). (B) Volcano plot illustrating the DESeq2 analysis of RNA-seq performed with HAP1 DNMT1 KO and DNMT1/L1TD1 DKO cells. Differentially expressed genes are plotted as DNMT1/L1TD1 DKO over DNMT1 KO (log2FC ≥1,

Article Snippet: DOI: https://doi.org/10.7554/eLife.96850 13 of 22 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody DNMT1 (H- 300) (polyclonal rabbit) Santa Cruz sc20701 RRID:AB_2293064 1:1000 Antibody ɣ-H2AX (monoclonal mouse) Millipore JBW301 RRID:AB_2847865 1:1000 Antibody Histone H3 C- term (polyclonal rabbit) Abcam Ab1791 RRID:AB_302613 1:5000 Antibody Cleaved caspase 3 (polyclonal rabbit) Cell Signaling 9661 RRID:AB_2341188 1:50 Antibody Anti- mouse HRP (polyclonal) Jackson Laboratories 115-035-008 RRID:AB_2313585 1:10,000 Antibody Anti- rabbit HRP (polyclonal) Jackson Laboratories 211- 032- 171 RRID:AB_2339149 1:10,000 Antibody Goat anti- Rabbit IgG (H+L) Alexa Fluor Plus 488 Invitrogen A32731 RRID:AB_2633280 1:500 Antibody Goat anti- Mouse IgG (H+L) Alexa Fluor Plus 546 Invitrogen A11030 RRID:AB_2737024 1:500 Sequence- based reagent hL1TD1 _f This paper PCR primers CTTA CCCT GG TAGC CGAC CT Sequence- based reagent hL1TD1 _r This paper PCR primers GGCT GGCA AA TTTT CTAA GG Sequence- based reagent hARMC1_f This paper PCR primers AGCT CTGG AG CGAA TTTA AGA Sequence- based reagent hARMC1_r This paper PCR primers GGCA GACA TC CCTG ATCC TG Sequence- based reagent hYY2_f This paper PCR primers TCCC GGAT AG CATT GAAG AC Sequence- based reagent hYY2_r This paper PCR primers TTGA CCTG CA TTTG CTTC TG Sequence- based reagent hORF1p_f This paper PCR primers AGTG CTTA AAG GAGC TGAT GG Sequence- based reagent hORF1p_r This paper PCR primers AACT GGAA GAA AGGG TATC AGC Commercial assay or kit CellTiter- Glo Luminiscent Cell Viability Assay Promega G7571 Commercial assay or kit Monarch RNA Cleanup Kit New England Biolabs T2047L Commercial assay or kit Qubit RNA High Sensitivity kit Thermo Fisher Scientific Q32852 Commercial assay or kit iScript cDNA synthesis Kit Bio- Rad 1708891 Commercial assay or kit Wizard Genomic DNA isolation kit Promega Commercial assay or kit EZ DNA Methylation Kit Zymo Research D5001 Software, algorithm DESeq2 Love et al., 2014 RRID:SCR_015687 Software, algorithm TEtranscript Jin et al., 2015 RRID:SCR_023208 Continued on next page Continued Kavaklioglu et al. eLife 2024;13:RP96850.

Techniques: Comparison, Mass Spectrometry, RNA Sequencing

Journal: eLife

Article Title: The domesticated transposon protein L1TD1 associates with its ancestor L1 ORF1p to promote LINE-1 retrotransposition

doi: 10.7554/elife.96850

Figure Lengend Snippet: Figure 4. L1TD1 promotes L1 retrotransposition. (A) Schematic representation of plasmids used for retrotransposition (figure modified from Kopera et al., 2016 and generated with BioRender.com). The pJJ101/L1.3 construct contains the full-length human L1.3 element with a blasticidin deaminase gene (mblast) inserted in antisense within the 3’UTR. The mblast gene is disrupted by an intron and mblast expression occurs only when L1 transcript is expressed, reverse transcribed, and inserted into the genome. The pJJ105/L1.3 plasmid contains a mutation in the reverse transcriptase (RT), resulting in defective retrotransposition. The backbone plasmid pCEP4 was used as additional negative control. The blasticidin deaminase gene containing plasmid pLenti6.2 was used as transfection/selection control. (B) Workflow of retrotransposition assay. DNMT1 KO and DNMT1/L1TD1 DKO cells were separately transfected with pJJ101 and control plasmids. Equal number of cells were seeded for each condition. Blasticidin selection (10 µg/ml) was started at day 4 and resistant colonies were counted on day 13. This panel was created using BioRender.com. (C) Bar graph showing the average number of retrotransposition events per 106 cells seeded in three independent experiments. Blasticidin-resistant colonies in pLenti6.2 transfected cells were used for normalization. Statistical significance was determined using unpaired t-test. All data in the figure are shown as a mean of ± SD of three independent experiments, ****p≤0.0001. (D) Representative pictures of bromophenol blue stainings of blasticidin-resistant colonies for each genotype and each transfection.

Article Snippet: DOI: https://doi.org/10.7554/eLife.96850 13 of 22 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody DNMT1 (H- 300) (polyclonal rabbit) Santa Cruz sc20701 RRID:AB_2293064 1:1000 Antibody ɣ-H2AX (monoclonal mouse) Millipore JBW301 RRID:AB_2847865 1:1000 Antibody Histone H3 C- term (polyclonal rabbit) Abcam Ab1791 RRID:AB_302613 1:5000 Antibody Cleaved caspase 3 (polyclonal rabbit) Cell Signaling 9661 RRID:AB_2341188 1:50 Antibody Anti- mouse HRP (polyclonal) Jackson Laboratories 115-035-008 RRID:AB_2313585 1:10,000 Antibody Anti- rabbit HRP (polyclonal) Jackson Laboratories 211- 032- 171 RRID:AB_2339149 1:10,000 Antibody Goat anti- Rabbit IgG (H+L) Alexa Fluor Plus 488 Invitrogen A32731 RRID:AB_2633280 1:500 Antibody Goat anti- Mouse IgG (H+L) Alexa Fluor Plus 546 Invitrogen A11030 RRID:AB_2737024 1:500 Sequence- based reagent hL1TD1 _f This paper PCR primers CTTA CCCT GG TAGC CGAC CT Sequence- based reagent hL1TD1 _r This paper PCR primers GGCT GGCA AA TTTT CTAA GG Sequence- based reagent hARMC1_f This paper PCR primers AGCT CTGG AG CGAA TTTA AGA Sequence- based reagent hARMC1_r This paper PCR primers GGCA GACA TC CCTG ATCC TG Sequence- based reagent hYY2_f This paper PCR primers TCCC GGAT AG CATT GAAG AC Sequence- based reagent hYY2_r This paper PCR primers TTGA CCTG CA TTTG CTTC TG Sequence- based reagent hORF1p_f This paper PCR primers AGTG CTTA AAG GAGC TGAT GG Sequence- based reagent hORF1p_r This paper PCR primers AACT GGAA GAA AGGG TATC AGC Commercial assay or kit CellTiter- Glo Luminiscent Cell Viability Assay Promega G7571 Commercial assay or kit Monarch RNA Cleanup Kit New England Biolabs T2047L Commercial assay or kit Qubit RNA High Sensitivity kit Thermo Fisher Scientific Q32852 Commercial assay or kit iScript cDNA synthesis Kit Bio- Rad 1708891 Commercial assay or kit Wizard Genomic DNA isolation kit Promega Commercial assay or kit EZ DNA Methylation Kit Zymo Research D5001 Software, algorithm DESeq2 Love et al., 2014 RRID:SCR_015687 Software, algorithm TEtranscript Jin et al., 2015 RRID:SCR_023208 Continued on next page Continued Kavaklioglu et al. eLife 2024;13:RP96850.

Techniques: Modification, Generated, Construct, Expressing, Reverse Transcription, Plasmid Preparation, Mutagenesis, Negative Control, Transfection, Selection, Control

Journal: Advanced Science

Article Title: PRMT5 Enables Robust STAT3 Activation via Arginine Symmetric Dimethylation of SMAD7

doi: 10.1002/advs.202003047

Figure Lengend Snippet: PRMT5 potentiates STAT3 activation via Smad7. A) PRMT5 depletion dampens endogenous STAT3 activation in A549 cells. A549 cells stably expressing shPRMT5‐1 or shPRMT5‐2 or Control (shCtrl) were harvested and analyzed by using western blotting with indicated antibodies. B) Knockdown of PRMT5 attenuates IL‐6‐induced STAT3 phosphorylation in MCF10A cells. MCF10A cells were transfected with 40 pm siRNA against PRMT5. 36 h later, cells were treated with IL‐6 (10 ng mL −1 ) for the indicated time and harvested for western blotting analysis with appropriate antibodies. C) PRMT5 inhibition attenuates endogenous activation of STAT3 in H358 cells. H358 cells were treated with 20 × 10 −6 m of PRMT5 inhibitors EPZ015666 or GSK591 for the indicated time. Cell lysates were collected and subject to western blotting analysis. SDMA indicates global arginine di‐methylation. D) Smad7 potentiates STAT3 activation in A549 cells. A549 cells stably expressing FLAG‐GFP or FLAG‐Smad7 were harvested and subject to Western blotting analysis using appropriate antibodies. E) Stable knockdown of Smad7 dampens endogenous STAT3 activation in A549 cells. A549 cells stably expressing shSmad7 or shCtrl were harvested and subject to western blotting analysis using appropriate antibodies. F) Smad7 depletion dampens IL‐6‐induced STAT3 activation in MCF7 cells. Cells were transfected with siSmad7 (40 pm) and treated with IL‐6 (10 ng mL −1 ) for the indicated time. Cells were harvested and analyzed by western blotting with appropriate antibodies. G) Smad7 depletion dampens IL‐6‐induced STAT3 activation in MCF10A cells. Cell transfection, treatment, and Western blotting were done as described in Panel F. H) PRMT5 potentiates STAT3 activation dependent of Smad7. MCF10A cells were transduced with lentiviral particles expressing HA‐PRMT5 or HA‐G367A/R368A. After 24 h, cells were transfected with 40 pm siSmad7. 12 h later, cells were stimulated with IL‐6 (2 ng mL −1 ) for the indicated time. Cell lysates were harvested and subject to Western blotting analysis using appropriate antibodies.

Article Snippet: Antibodies and their commercial sources are as follows: PRMT5 (ab109451) and gp130 (ab202850) from Abcam; p‐STAT3 (9145), STAT3 (9139), HA (3724) and sdme‐RG (13222) from Cell Signaling Technology; SYM10 (07‐412) from Merck millipore; FLAG (F3165), β ‐actin (A5441), mouse IgG (I5381), and rabbit IgG(I5006) from Sigma‐Aldrich (USA); MYC (sc‐40) from Santa Cruz Biotechnology; Survivin (A19663) and c‐MYC (A1309) from Abclonal.

Techniques: Activation Assay, Stable Transfection, Expressing, Western Blot, Transfection, Inhibition, Methylation, Transduction

Journal: Advanced Science

Article Title: PRMT5 Enables Robust STAT3 Activation via Arginine Symmetric Dimethylation of SMAD7

doi: 10.1002/advs.202003047

Figure Lengend Snippet: PRMT5 and MEP50 interact with Smad7. A) Smad7 interacts with PRMT5 and requires MEP50. HEK293T cells were transfected with HA‐PRMT5, FLAG‐Smad7, and MYC‐MEP50. Cell lysates were harvested and immunoprecipitated with HA antibody. The immunocomplexes and input were analyzed by using Western blotting analysis with indicated antibodies. B) Smad7 interacts with PRMT5 and MEP50 in vitro. Recombinant GST‐Smad7 or GST protein was produced and purified from Escherichia coli . MYC‐PRMT5 or G367A/R368A mutant together with MEP50 were expressed in HEK293T cells. In the GST pulldown assay, MYC‐PRMT5 or G367A/R368A proteins bound to GST proteins were retrieved with glutathione sepharose beads, and then analyzed by using Western blotting. C) PRMT5/MEP50 interacts with Smad7 in the MH2 domain. HEK293T cell transfection and Western blotting analysis were similarly done as described in Panel A. D) Smad7 interacts with endogenous PRMT5. Expression of SFB‐Smad7 was induced with or without 500 ng mL −1 Dox for 3 d in MCF10A‐tet‐on cells, and treated with 25 ng mL −1 IL‐6 for the indicated time. Cell lysates were harvested, precipitated with streptavidin beads, and analyzed by using Western blotting.

Article Snippet: Antibodies and their commercial sources are as follows: PRMT5 (ab109451) and gp130 (ab202850) from Abcam; p‐STAT3 (9145), STAT3 (9139), HA (3724) and sdme‐RG (13222) from Cell Signaling Technology; SYM10 (07‐412) from Merck millipore; FLAG (F3165), β ‐actin (A5441), mouse IgG (I5381), and rabbit IgG(I5006) from Sigma‐Aldrich (USA); MYC (sc‐40) from Santa Cruz Biotechnology; Survivin (A19663) and c‐MYC (A1309) from Abclonal.

Techniques: Transfection, Immunoprecipitation, Western Blot, In Vitro, Recombinant, Produced, Purification, Mutagenesis, GST Pulldown Assay, Expressing

Journal: Advanced Science

Article Title: PRMT5 Enables Robust STAT3 Activation via Arginine Symmetric Dimethylation of SMAD7

doi: 10.1002/advs.202003047

Figure Lengend Snippet: PRMT5 methylates Smad7 on R57. A) PRMT5 methylates Smad7. HEK293T cells were transfected with expression plasmids carrying MYC‐PRMT5/MEP50 and an SFB‐tagged construct, including gp130, JAK2, STAT3, Smad7, SHP2, and SOCS3. Cell lysates were harvested and precipitated with streptavidin beads. The retrieved complexes and input were analyzed by Western blotting with indicated antibodies. B) PRMT5 methylates the R57 residue on Smad7. HEK293T cells were transfected with MYC‐PRMT5/MEP50 and an SFB‐tagged Smad7 construct, i.e., wildtype Smad7 (WT) or a R‐to‐K substitution of Smad7 as indicated above the blots. Cell lysate was precipitated with streptavidin beads. Arginine di‐methylation of Smad7 was detected by Western blotting analysis. C) Mass spectrum of Smad7 Arg‐57 dimethylated peptide. Mass spectrometry identified Arg‐57 dimethylation of Smad7 in HEK293T cells expressing MYC‐PRMT5/MEP50 and SFB‐Smad7. Mass spectrometry profile of Smad7 sequence covering residue 47–64 is shown, and the dimethylated arginine side chains are indicated. D) PRMT5/MEP50 methylate Smad7, but not the R57K mutant. HEK293T cells were transfected MYC‐PRMT5/MEP50 and SFB‐Smad7 or Smad7 R57K mutant for 36 h. Cell lysates were harvested and immunoprecipitated with SYM10 antibody. The immunocomplexes and inputs were analyzed by Western blotting with indicated antibodies. E) PRMT5 methylates Smad7 in vitro. MYC‐PRMT5 or MYC‐G367A/R368A together with MYC‐MEP50 were immunopurified using anti‐MYC antibody from transfected HEK293T cells. Purified recombinant GST‐Smad7, GST‐Smad7 R57K mutant, and GST‐Smad4 were produced in E. coli . GST proteins and MYC‐PRMT5/MEP50 proteins were incubated in the presence of S‐adenosyl‐methionine to allow methylation reaction. Dimethylated Smad7 on R57 was detected by using Western blotting analysis.

Article Snippet: Antibodies and their commercial sources are as follows: PRMT5 (ab109451) and gp130 (ab202850) from Abcam; p‐STAT3 (9145), STAT3 (9139), HA (3724) and sdme‐RG (13222) from Cell Signaling Technology; SYM10 (07‐412) from Merck millipore; FLAG (F3165), β ‐actin (A5441), mouse IgG (I5381), and rabbit IgG(I5006) from Sigma‐Aldrich (USA); MYC (sc‐40) from Santa Cruz Biotechnology; Survivin (A19663) and c‐MYC (A1309) from Abclonal.

Techniques: Transfection, Expressing, Construct, Western Blot, Methylation, Mass Spectrometry, Sequencing, Mutagenesis, Immunoprecipitation, In Vitro, Purification, Recombinant, Produced, Incubation

Journal: Advanced Science

Article Title: PRMT5 Enables Robust STAT3 Activation via Arginine Symmetric Dimethylation of SMAD7

doi: 10.1002/advs.202003047

Figure Lengend Snippet: Arg methylation enhances Smad7 binding to gp130. A) Smad7 methylation increases its association with gp130. HEK293T cells were transfected with SFB‐Smad7 or Smad7 R57K mutant and HA‐gp130, together with MYC‐PRMT5/MEP50. Cell lysates were harvested and immunoprecipitated with Streptavidin beads. Western blotting analysis was done with appropriate antibodies. B) PRMT5 depletion blocks Smad7 methylation and its interaction with endogenous gp130. A549 tet‐on cells expressing SFB‐Smad7 were cultured with or without 1 µg mL −1 Dox for 3 d and then transfected with 40 × 10 −12 m siPRMT5. Cell lysates were harvested and immunoprecipitated with streptavidin beads. Endogenous gp130 was detected from the immunoprecipitates by using Western blotting analysis. C) Methylated Smad7 binds more tightly to gp130. HEK293T cells were transfected with indicated expression plasmids for MYC‐PRMT5, MYC‐G367A/R368A, and MYC‐MEP50 as well as SFB‐Smad7 or SFB‐R57K. Dimethylated Smad7 was immunopurified using SYM10 antibody, while total Smad7 was retrieved using an‐FLAG antibody. Bacterially expressed GST‐gp130‐ICD was purified using glutathione‐sepharose and eluted with elution buffer (10 × 10 −3 m glutathione, pH 8.0). In the in vitro binding experiments for evaluating the Smad7‐gp130 interaction, recombinant GST‐gp130‐ICD was added to the immunopurified Smad7. gp130‐ICD binding to immobilized Smad7 was analyzed by using Western blotting. D) Unmethylatable Smad7 R57K mutant loses its ability to potentiate STAT3 activation. MCF10A tet‐on cells stably expressing SFB‐Smad7 or Smad7 R57K were induced with 10 ng mL −1 Dox for 3 d, and treated with indicated concentrations of IL‐6. Cell lysates were collected and subject to Western blotting analysis.

Article Snippet: Antibodies and their commercial sources are as follows: PRMT5 (ab109451) and gp130 (ab202850) from Abcam; p‐STAT3 (9145), STAT3 (9139), HA (3724) and sdme‐RG (13222) from Cell Signaling Technology; SYM10 (07‐412) from Merck millipore; FLAG (F3165), β ‐actin (A5441), mouse IgG (I5381), and rabbit IgG(I5006) from Sigma‐Aldrich (USA); MYC (sc‐40) from Santa Cruz Biotechnology; Survivin (A19663) and c‐MYC (A1309) from Abclonal.

Techniques: Methylation, Binding Assay, Transfection, Mutagenesis, Immunoprecipitation, Western Blot, Expressing, Cell Culture, Purification, In Vitro, Recombinant, Activation Assay, Stable Transfection

Journal: Advanced Science

Article Title: PRMT5 Enables Robust STAT3 Activation via Arginine Symmetric Dimethylation of SMAD7

doi: 10.1002/advs.202003047

Figure Lengend Snippet: PRMT5 promotes STAT3 transcriptional and growth‐promoting responses. A) PRMT5 inhibition attenuates CDC25C expression in A549 cells. EPZ015666 or GSK591 (20 × 10 −6 m ) were added to A549 cells for 48 h. Cell lysates were harvested and analyzed by using qRT‐PCR to examine CDC25C mRNA levels. Data are shown as mean ± SD; n = 3. *** P < 0.001. B) PRMT5 inhibition attenuates CCNB1 expression in A549 cells. Cell treatment, harvest, and qRT‐PCR analysis were done as described in Panel A. Data are shown as mean ± SD; n = 3. *** P < 0.001. C) PRMT5 deficiency disables IL‐6/STAT3 responsiveness. GSEA showed that downregulated genes in PRMT5‐depleted A549 cells (shPRMT5‐2) were highly enriched in the IL‐6/STAT3 signaling gene set. Red, upregulated genes; blue, downregulated genes. NES = ‐1.73, FDR q value = 0.002. D) Heatmap showing expression levels (log 2 FPKM; left) and relative expression changes (log 2 (shPRMT5‐2/shCtrl); right) of the IL‐6/STAT3 signaling genes. E) Depletion of PRMT5 reduces DNA synthesis. A549 cells stably expressing shPRMT5‐1 or shPRMT5‐2 or Control (shCtrl) were subject to EdU staining to determine DNA incorporating rate (RiboBio),20x. F) Statistic analysis of the result in panel E. Data are shown as mean ± SD; n = 3. 0.01 < * P < 0.05. G) Inhibition of PRMT5 attenuates invasiveness in A549 cells. A549 cells were treated with PRMT5 inhibitors EPZ015666 or GSK591 (20 × 10 −6 m ) for 2 d, starved overnight in FBS‐free medium. 1 × 10 5 cells were plated in a transwell chamber and stained with crystal violet after 12 h. Purple color indicates crystal violet staining of the invaded cell population. H) PRMT5 depletion blocks colony formation. A549 stable cells were subject to crystal violet staining and photography.

Article Snippet: Antibodies and their commercial sources are as follows: PRMT5 (ab109451) and gp130 (ab202850) from Abcam; p‐STAT3 (9145), STAT3 (9139), HA (3724) and sdme‐RG (13222) from Cell Signaling Technology; SYM10 (07‐412) from Merck millipore; FLAG (F3165), β ‐actin (A5441), mouse IgG (I5381), and rabbit IgG(I5006) from Sigma‐Aldrich (USA); MYC (sc‐40) from Santa Cruz Biotechnology; Survivin (A19663) and c‐MYC (A1309) from Abclonal.

Techniques: Inhibition, Expressing, Quantitative RT-PCR, DNA Synthesis, Stable Transfection, Staining

Journal: Advanced Science

Article Title: PRMT5 Enables Robust STAT3 Activation via Arginine Symmetric Dimethylation of SMAD7

doi: 10.1002/advs.202003047

Figure Lengend Snippet: PRMT5 promotes lung tumorigenesis. A) Depletion of PRMT5 attenuates tumorigenesis. LLC cells stably expressing shControl or mouse sh‐mPRMT5‐1 or sh‐mPRMT5‐2 were subcutaneously injected into female nude mice. Ten days after implantation, tumors were dissected and photographed. B) Measurement of tumor weight in Panel A. Data are shown as mean ± SD; n = 5 for each group. 0.01 < * P < 0.05. C) PRMT5 depletion impairs STAT3 signaling in tumors. Tumor samples were analyzed by Western blotting to examine phosphorylated STAT3 (p‐STAT3) and STAT3 target gene products such as c‐Myc and Survivin. D) PRMT5 is highly expressed in nonsmall cell lung cancer tissues (NSCLC). NSCLC tissue microarray (Alenabio) was subject to immunohistochemistry (Servicebio) using PRMT5 antibody. E) Statistic analysis of IHC score in Panel D. Statistical analysis was performed using a two‐tailed Student's t ‐test. Data are shown as mean ± SD. Lung cancer samples = 45. Normal lung tissue samples = 55. *** P < 0.001. F) A working model for PRMT5‐mediated STAT3 activation.

Article Snippet: Antibodies and their commercial sources are as follows: PRMT5 (ab109451) and gp130 (ab202850) from Abcam; p‐STAT3 (9145), STAT3 (9139), HA (3724) and sdme‐RG (13222) from Cell Signaling Technology; SYM10 (07‐412) from Merck millipore; FLAG (F3165), β ‐actin (A5441), mouse IgG (I5381), and rabbit IgG(I5006) from Sigma‐Aldrich (USA); MYC (sc‐40) from Santa Cruz Biotechnology; Survivin (A19663) and c‐MYC (A1309) from Abclonal.

Techniques: Stable Transfection, Expressing, Injection, Western Blot, Microarray, Immunohistochemistry, Two Tailed Test, Activation Assay

Journal: Investigative Ophthalmology & Visual Science

Article Title: Transgenic Expression of Constitutively Active RAC1 Disrupts Mouse Rod Morphogenesis

doi: 10.1167/iovs.13-13649

Figure Lengend Snippet: List of Primary Antibodies Used in Immunohistochemistry

Article Snippet: Examination was done on three to four animals for each genotype per age group (P6, P11, or P24). table ft1 table-wrap mode="anchored" t5 caption a7 Name Target Host Dilution Product Source MYC-Tag CA RAC1 Mouse 1:2,000 2276; Cell Signaling Technology, Inc., Danvers, MA, USA Rabbit 1:400 2272; Cell Signaling Technology, Inc. Cone arrestin Cone Rabbit 1:10,000 AB15282; Millipore, Temecula, CA, USA Rhodopsin Rod Mouse 1:10,000 O 4886; Sigma-Aldrich, St. Louis, MO, USA Retinoschisin IS marker Rabbit 1:1,000 Takada et al. 57 Synaptophysin Presynaptic marker Mouse 1:200 S 5768; Sigma-Aldrich PKCα Rod bipolar cell Rabbit 1:1,000 P 4334; Sigma-Aldrich PH3 Mitosis marker Rabbit 1:100 06-570; Millipore Ki67 Progenitor cell marker Mouse 1:400 556003; BD Biosciences, San Jose, CA, USA BrdU DNA synthesis marker Rat 1:100 Ab6326; Abcam, Inc., Cambridge, MA, USA PAR6 Polarity protein Rabbit 1:100 Sc-67393; Santa Cruz Biotechnology, Dallas, TX, USA PAR3 Polarity protein Rabbit 1:50 11085-1-AP; Proteintech Group, Chicago, IL, USA aPKCλ Polarity protein Rabbit 1:100 Sc-11399; Santa Cruz Biotechnology β-catenin OLM marker Mouse 1:300 610153; BD Biosciences DIC Dynein intermediate chain Mouse 1:400 MAB1618; Millipore Open in a separate window List of Primary Antibodies Used in Immunohistochemistry

Techniques: Marker, DNA Synthesis

Journal: Investigative Ophthalmology & Visual Science

Article Title: Transgenic Expression of Constitutively Active RAC1 Disrupts Mouse Rod Morphogenesis

doi: 10.1167/iovs.13-13649

Figure Lengend Snippet: Constitutively active RAC1 expression in transgenic mice. (A) Western blotting with anti-RAC1 (endogenous RAC1 and transgenic CA RAC1) and anti-MYC-Tag (transgenic CA RAC1-specific) antibodies at P4, P6, and P11 indicated that CA RAC1 expression in the retinas increased with age in both LO and HI transgenic lines. Constitutively active RAC1 protein bands are yellow due to an overlap of anti-RAC1 (red) and anti-MYC-Tag (green) antibodies. (B) Fluorescence values of the CA RAC1 band in each sample at P6 were normalized to fluorescence values of corresponding endogenous RAC1 band. Quantification of CA RAC1 expression is represented as a percentage of endogenous RAC1 levels in CA RAC1 retinas, shown by the bar graph (mean ± SEM, n = 2).

Article Snippet: Examination was done on three to four animals for each genotype per age group (P6, P11, or P24). table ft1 table-wrap mode="anchored" t5 caption a7 Name Target Host Dilution Product Source MYC-Tag CA RAC1 Mouse 1:2,000 2276; Cell Signaling Technology, Inc., Danvers, MA, USA Rabbit 1:400 2272; Cell Signaling Technology, Inc. Cone arrestin Cone Rabbit 1:10,000 AB15282; Millipore, Temecula, CA, USA Rhodopsin Rod Mouse 1:10,000 O 4886; Sigma-Aldrich, St. Louis, MO, USA Retinoschisin IS marker Rabbit 1:1,000 Takada et al. 57 Synaptophysin Presynaptic marker Mouse 1:200 S 5768; Sigma-Aldrich PKCα Rod bipolar cell Rabbit 1:1,000 P 4334; Sigma-Aldrich PH3 Mitosis marker Rabbit 1:100 06-570; Millipore Ki67 Progenitor cell marker Mouse 1:400 556003; BD Biosciences, San Jose, CA, USA BrdU DNA synthesis marker Rat 1:100 Ab6326; Abcam, Inc., Cambridge, MA, USA PAR6 Polarity protein Rabbit 1:100 Sc-67393; Santa Cruz Biotechnology, Dallas, TX, USA PAR3 Polarity protein Rabbit 1:50 11085-1-AP; Proteintech Group, Chicago, IL, USA aPKCλ Polarity protein Rabbit 1:100 Sc-11399; Santa Cruz Biotechnology β-catenin OLM marker Mouse 1:300 610153; BD Biosciences DIC Dynein intermediate chain Mouse 1:400 MAB1618; Millipore Open in a separate window List of Primary Antibodies Used in Immunohistochemistry

Techniques: Expressing, Transgenic Assay, Western Blot, Fluorescence

Journal: Investigative Ophthalmology & Visual Science

Article Title: Transgenic Expression of Constitutively Active RAC1 Disrupts Mouse Rod Morphogenesis

doi: 10.1167/iovs.13-13649

Figure Lengend Snippet: Time course of abnormal outer nuclear layer (ONL) lamination in CA RAC1 retinas during retinal development. (A) Retinal sections from CA RAC1 (LO and HI) and nontransgenic WT (Control) littermates at several postnatal times (P4, P6, P11, P21, and P3m) stained with hematoxylin and eosin showed progressive defects in the ONL lamination of CA RAC1 retinas. At P4, CA RAC1 retinas (b, c) had apparently normal neuroblastic layers (NBL). At P6, the IS appeared in both control (d) and CA RAC1 (e, f) retinas, but some nuclei were mislocalized into the space between the RPE and IS layer in CA RAC1 retinas (e, f) (black arrows). The OPL formed closer to the apical margin of the ONL than in the control, leading to a thin ONL, particularly in the CA RAC1 HI line (f). At P11, additional nuclei were displaced into the presumptive subretinal space adjacent to the RPE in both transgenic lines (h, i). ONL nuclei aberrantly formed retinal folds or whorls in the central retinal region. Yellow arrows indicate the areas bridging ONL to INL with incomplete separation by the OPL. By P21, both LO and HI lines had fewer ONL nuclei (k, l), compared with the WT control (j). By 3 months (P3m), the ONL was reduced to a single layer of nuclei in the HI line (o) and two to three rows in the LO line (n). Scale bar: 50 μm. (B) Retinal sections of nontransgenic control (Control) and CA RAC1 littermates at P24 with TUNEL assay showed increased apoptosis signals (red) at ONL in CA RAC1 retinas (q), compared to the control (p). Scale bar: 20 μm. IPL, inner plexiform layer, GCL, ganglion cell layer.

Article Snippet: Examination was done on three to four animals for each genotype per age group (P6, P11, or P24). table ft1 table-wrap mode="anchored" t5 caption a7 Name Target Host Dilution Product Source MYC-Tag CA RAC1 Mouse 1:2,000 2276; Cell Signaling Technology, Inc., Danvers, MA, USA Rabbit 1:400 2272; Cell Signaling Technology, Inc. Cone arrestin Cone Rabbit 1:10,000 AB15282; Millipore, Temecula, CA, USA Rhodopsin Rod Mouse 1:10,000 O 4886; Sigma-Aldrich, St. Louis, MO, USA Retinoschisin IS marker Rabbit 1:1,000 Takada et al. 57 Synaptophysin Presynaptic marker Mouse 1:200 S 5768; Sigma-Aldrich PKCα Rod bipolar cell Rabbit 1:1,000 P 4334; Sigma-Aldrich PH3 Mitosis marker Rabbit 1:100 06-570; Millipore Ki67 Progenitor cell marker Mouse 1:400 556003; BD Biosciences, San Jose, CA, USA BrdU DNA synthesis marker Rat 1:100 Ab6326; Abcam, Inc., Cambridge, MA, USA PAR6 Polarity protein Rabbit 1:100 Sc-67393; Santa Cruz Biotechnology, Dallas, TX, USA PAR3 Polarity protein Rabbit 1:50 11085-1-AP; Proteintech Group, Chicago, IL, USA aPKCλ Polarity protein Rabbit 1:100 Sc-11399; Santa Cruz Biotechnology β-catenin OLM marker Mouse 1:300 610153; BD Biosciences DIC Dynein intermediate chain Mouse 1:400 MAB1618; Millipore Open in a separate window List of Primary Antibodies Used in Immunohistochemistry

Techniques: Control, Staining, Transgenic Assay, TUNEL Assay

Journal: Investigative Ophthalmology & Visual Science

Article Title: Transgenic Expression of Constitutively Active RAC1 Disrupts Mouse Rod Morphogenesis

doi: 10.1167/iovs.13-13649

Figure Lengend Snippet: Mislocalization of rod and cone photoreceptors in CA RAC1 retinas. Retinal sections of nontransgenic control and CA RAC1 mice were double immunostained with antibodies against MYC-Tag (green, CA RAC1) and rhodopsin (red) (A) or cone arrestin (red) (B). Retinal nuclei were labeled with DAPI (gray or blue). (A) Representative DAPI and merged images are shown. Mislocalized cells in the subretinal space in CA RAC1 retinas at P6 ([c–e], boxed regions) and at P11 ([h–j], boxed regions) were positive for CA RAC1 (green) and rhodopsin (red) double immunostaining (higher magnification images of boxed regions in [d] and [i] are shown in [e] and [j], respectively), indicating that these mislocalized cells were rod photoreceptors. Many more cells double-labeled with CA RAC1 (green) and rhodopsin (red) antibodies were mislocalized in the INL at P6 (d) and P11 (i), compared to the control (b, g). (a–d, f–i) Scale bar: 20 μm; (e, j) scale bar: 5 μm. (B) Merged immunostaining images of CA RAC1 (green) and cone arrestin (red) are shown in the control (k) and CA RAC1 (l) retinas at P24. Cone arrestin–positive somas were located at the apical surface of the ONL in the control, whereas cone arrestin–stained cell bodies were mislocalized in the CA RAC1 retina ([l], white arrows). (k, l) Scale bar: 10 μm. A magnified region of the white box in (l) is shown in (m, n). Dotted outlines in (m, n) display the location of cone arrestin–positive cell bodies, indicating that the cone cells were negative for CA RAC1 staining in CA RAC1 retinas. Scale bar: 2.5 μm.

Article Snippet: Examination was done on three to four animals for each genotype per age group (P6, P11, or P24). table ft1 table-wrap mode="anchored" t5 caption a7 Name Target Host Dilution Product Source MYC-Tag CA RAC1 Mouse 1:2,000 2276; Cell Signaling Technology, Inc., Danvers, MA, USA Rabbit 1:400 2272; Cell Signaling Technology, Inc. Cone arrestin Cone Rabbit 1:10,000 AB15282; Millipore, Temecula, CA, USA Rhodopsin Rod Mouse 1:10,000 O 4886; Sigma-Aldrich, St. Louis, MO, USA Retinoschisin IS marker Rabbit 1:1,000 Takada et al. 57 Synaptophysin Presynaptic marker Mouse 1:200 S 5768; Sigma-Aldrich PKCα Rod bipolar cell Rabbit 1:1,000 P 4334; Sigma-Aldrich PH3 Mitosis marker Rabbit 1:100 06-570; Millipore Ki67 Progenitor cell marker Mouse 1:400 556003; BD Biosciences, San Jose, CA, USA BrdU DNA synthesis marker Rat 1:100 Ab6326; Abcam, Inc., Cambridge, MA, USA PAR6 Polarity protein Rabbit 1:100 Sc-67393; Santa Cruz Biotechnology, Dallas, TX, USA PAR3 Polarity protein Rabbit 1:50 11085-1-AP; Proteintech Group, Chicago, IL, USA aPKCλ Polarity protein Rabbit 1:100 Sc-11399; Santa Cruz Biotechnology β-catenin OLM marker Mouse 1:300 610153; BD Biosciences DIC Dynein intermediate chain Mouse 1:400 MAB1618; Millipore Open in a separate window List of Primary Antibodies Used in Immunohistochemistry

Techniques: Control, Labeling, Double Immunostaining, Immunostaining, Staining

Journal: Investigative Ophthalmology & Visual Science

Article Title: Transgenic Expression of Constitutively Active RAC1 Disrupts Mouse Rod Morphogenesis

doi: 10.1167/iovs.13-13649

Figure Lengend Snippet: Characterization of mislocalized rod cells in the presumptive subretinal space adjacent to the RPE in CA RAC1 retinas. (A) Rod photoreceptor IS and OS were immunostained with antibodies against retinoschisin (RS, green) and rhodopsin (red), respectively, at P11 and P21. In the CA RAC1 retina (b, d), the mislocalized photoreceptors in the subretinal space (white arrows) did not show any normally oriented RS (green) and rhodopsin (red) labeling. Scale bar: 20 μm. (B) TEMs of photoreceptor cells are shown for nontransgenic control (e) and CA RAC1 (f) retinas at P21. No IS/OS structure was found between mislocalized cells (R, C) and the RPE (f). Note that mislocalized cells adjacent to the RPE included rods (R, large clumps of heterochromatin typical of rod nuclei) and cones (C, one or more small clumps of heterochromatin surrounded by a large amount of lighter staining euchromatin characteristic of cone nuclei). Scale bars: 5 μm. (C) Photoreceptor synaptic terminals were detected by immunostaining with synaptophysin (SYN, red), a presynaptic marker, and protein kinase C alpha (PKCα, green), a rod bipolar cell marker. In comparison to the nontransgenic control (g) in which a continuous band of staining with SYN and PKCα was seen across OPL synaptic terminals, the CA RAC1 retina showed a fragmented staining pattern (h). In the CA RAC1 retina, ectopic staining of SYN was observed around apically mislocalized cells that was not associated with PKCα-stained rod bipolar cells ([h], white arrows) and PKCα-positive bipolar cell process protruded into the ONL to contact synaptic terminals ([h], yellow arrow). Scale bar: 10 μm. (D) TEM images of synaptic ribbons (SR) are shown in the nontransgenic control ([i], red arrow) and in a mislocalized cell adjacent to the RPE in the CA RAC1 retina ([j], red arrow). A higher magnification image of SR in (j) is shown in (k) (red arrow). Note that, in the CA RAC1 retina, a displaced ribbon tethered multiple vesicles and pointed toward the RPE. No horizontal (H) or bipolar (B) cells were found around the ribbon. N, nucleus. (i) Scale bar: 500 nm; (j) scale bar: 2 μm; (k) scale bar: 100 nm.

Article Snippet: Examination was done on three to four animals for each genotype per age group (P6, P11, or P24). table ft1 table-wrap mode="anchored" t5 caption a7 Name Target Host Dilution Product Source MYC-Tag CA RAC1 Mouse 1:2,000 2276; Cell Signaling Technology, Inc., Danvers, MA, USA Rabbit 1:400 2272; Cell Signaling Technology, Inc. Cone arrestin Cone Rabbit 1:10,000 AB15282; Millipore, Temecula, CA, USA Rhodopsin Rod Mouse 1:10,000 O 4886; Sigma-Aldrich, St. Louis, MO, USA Retinoschisin IS marker Rabbit 1:1,000 Takada et al. 57 Synaptophysin Presynaptic marker Mouse 1:200 S 5768; Sigma-Aldrich PKCα Rod bipolar cell Rabbit 1:1,000 P 4334; Sigma-Aldrich PH3 Mitosis marker Rabbit 1:100 06-570; Millipore Ki67 Progenitor cell marker Mouse 1:400 556003; BD Biosciences, San Jose, CA, USA BrdU DNA synthesis marker Rat 1:100 Ab6326; Abcam, Inc., Cambridge, MA, USA PAR6 Polarity protein Rabbit 1:100 Sc-67393; Santa Cruz Biotechnology, Dallas, TX, USA PAR3 Polarity protein Rabbit 1:50 11085-1-AP; Proteintech Group, Chicago, IL, USA aPKCλ Polarity protein Rabbit 1:100 Sc-11399; Santa Cruz Biotechnology β-catenin OLM marker Mouse 1:300 610153; BD Biosciences DIC Dynein intermediate chain Mouse 1:400 MAB1618; Millipore Open in a separate window List of Primary Antibodies Used in Immunohistochemistry

Techniques: Labeling, Control, Staining, Immunostaining, Marker, Comparison

Journal: Investigative Ophthalmology & Visual Science

Article Title: Transgenic Expression of Constitutively Active RAC1 Disrupts Mouse Rod Morphogenesis

doi: 10.1167/iovs.13-13649

Figure Lengend Snippet: Localization of retinal progenitor cells and migration of newly differentiated photoreceptors in CA RAC1 retinas. (A) Retinal sections of CA RAC1 and nontransgenic littermates (Control) at P6 immunostained with the antibody against pH3 (red, [a, b, e, f]) or Ki-67 (red, [c, d, g, h]). Nuclei were counterstained with DAPI. Images of central and periphery regions show similar staining patterns of pH3 and Ki-67 in control (a–d) and CA RAC1 (e–h) retinas. Note that no pH3- or Ki-67-positive labeling was detected within the mislocalized nuclei adjacent to the RPE ([e] and [g], white arrows). (B) The movement of BrdU-labeled cells in CA RAC1 retinas. Immunofluorescence images of BrdU-labeled cells (red) in nontransgenic control (i–k) and CA RAC1 (l–n) retinas are shown at 3 hours (P1), 4 days (P5), and 10 days (P11) after BrdU injection at P1. In CA RAC1 retinas, more mislocalized cells in the subretinal space (indicated by white arrows in [m] and [n]) were labeled by BrdU (red) at P11 (n) than at P5 (m). (o) Double staining of BrdU (red, stains the nucleus) and rhodopsin (green, stains the plasma membrane) indicates that BrdU-positive mislocalized cells in the subretinal space also stained for rhodopsin. The inset in (o) is higher magnification of the white box region. (i–o) Scale bar: 20 μm. Scale bar in the inset: 5 μm.

Article Snippet: Examination was done on three to four animals for each genotype per age group (P6, P11, or P24). table ft1 table-wrap mode="anchored" t5 caption a7 Name Target Host Dilution Product Source MYC-Tag CA RAC1 Mouse 1:2,000 2276; Cell Signaling Technology, Inc., Danvers, MA, USA Rabbit 1:400 2272; Cell Signaling Technology, Inc. Cone arrestin Cone Rabbit 1:10,000 AB15282; Millipore, Temecula, CA, USA Rhodopsin Rod Mouse 1:10,000 O 4886; Sigma-Aldrich, St. Louis, MO, USA Retinoschisin IS marker Rabbit 1:1,000 Takada et al. 57 Synaptophysin Presynaptic marker Mouse 1:200 S 5768; Sigma-Aldrich PKCα Rod bipolar cell Rabbit 1:1,000 P 4334; Sigma-Aldrich PH3 Mitosis marker Rabbit 1:100 06-570; Millipore Ki67 Progenitor cell marker Mouse 1:400 556003; BD Biosciences, San Jose, CA, USA BrdU DNA synthesis marker Rat 1:100 Ab6326; Abcam, Inc., Cambridge, MA, USA PAR6 Polarity protein Rabbit 1:100 Sc-67393; Santa Cruz Biotechnology, Dallas, TX, USA PAR3 Polarity protein Rabbit 1:50 11085-1-AP; Proteintech Group, Chicago, IL, USA aPKCλ Polarity protein Rabbit 1:100 Sc-11399; Santa Cruz Biotechnology β-catenin OLM marker Mouse 1:300 610153; BD Biosciences DIC Dynein intermediate chain Mouse 1:400 MAB1618; Millipore Open in a separate window List of Primary Antibodies Used in Immunohistochemistry

Techniques: Migration, Control, Staining, Labeling, Immunofluorescence, Injection, Double Staining, Clinical Proteomics, Membrane

Journal: Investigative Ophthalmology & Visual Science

Article Title: Transgenic Expression of Constitutively Active RAC1 Disrupts Mouse Rod Morphogenesis

doi: 10.1167/iovs.13-13649

Figure Lengend Snippet: Disrupted localization of the PAR6 complex in the CA RAC1 retina. (A) Retinal extracts (Extracts) of CA RAC1 and nontransgenic WT (Control) littermates at P11 were immunoprecipitated using either anti-RAC1 (endogenous RAC1 and transgenic CA RAC1, RAC1-IP) or anti-MYC-Tag (transgenic CA RAC1-specific, MYC-IP) antibody and control IgG (IgG-IP). Co-immunoprecipitated proteins were analyzed by Western blotting. PAR6 was co-IP in both RAC1-IP and MYC-IP immunocomplexes of CA RAC1 retinal extracts, but only in RAC1-IP immunocomplex of the control. (B) Retinal sections of CA RAC1 mice at P11 were double immunostained with antibodies against MYC-Tag (green, CA RAC1) and PAR6 (red). Nuclei were labeled with DAPI (blue). Merged images indicate that CA RAC1 colocalized with PAR6. Scale bar: 20 μm. (C) Retinal sections of nontransgenic control and CA RAC1 littermates at P11 were immunostained with antibodies against components of the PAR6 complex (PAR6, aPKCλ, and PAR3) and β-catenin, an OLM marker. Immunostaining pattern of these proteins was disrupted and fragmented in CA RAC1 retinas. Scale bar: 20 μm.

Article Snippet: Examination was done on three to four animals for each genotype per age group (P6, P11, or P24). table ft1 table-wrap mode="anchored" t5 caption a7 Name Target Host Dilution Product Source MYC-Tag CA RAC1 Mouse 1:2,000 2276; Cell Signaling Technology, Inc., Danvers, MA, USA Rabbit 1:400 2272; Cell Signaling Technology, Inc. Cone arrestin Cone Rabbit 1:10,000 AB15282; Millipore, Temecula, CA, USA Rhodopsin Rod Mouse 1:10,000 O 4886; Sigma-Aldrich, St. Louis, MO, USA Retinoschisin IS marker Rabbit 1:1,000 Takada et al. 57 Synaptophysin Presynaptic marker Mouse 1:200 S 5768; Sigma-Aldrich PKCα Rod bipolar cell Rabbit 1:1,000 P 4334; Sigma-Aldrich PH3 Mitosis marker Rabbit 1:100 06-570; Millipore Ki67 Progenitor cell marker Mouse 1:400 556003; BD Biosciences, San Jose, CA, USA BrdU DNA synthesis marker Rat 1:100 Ab6326; Abcam, Inc., Cambridge, MA, USA PAR6 Polarity protein Rabbit 1:100 Sc-67393; Santa Cruz Biotechnology, Dallas, TX, USA PAR3 Polarity protein Rabbit 1:50 11085-1-AP; Proteintech Group, Chicago, IL, USA aPKCλ Polarity protein Rabbit 1:100 Sc-11399; Santa Cruz Biotechnology β-catenin OLM marker Mouse 1:300 610153; BD Biosciences DIC Dynein intermediate chain Mouse 1:400 MAB1618; Millipore Open in a separate window List of Primary Antibodies Used in Immunohistochemistry

Techniques: Control, Immunoprecipitation, Transgenic Assay, Western Blot, Co-Immunoprecipitation Assay, Labeling, Marker, Immunostaining

Journal: Investigative Ophthalmology & Visual Science

Article Title: Transgenic Expression of Constitutively Active RAC1 Disrupts Mouse Rod Morphogenesis

doi: 10.1167/iovs.13-13649

Figure Lengend Snippet: Constitutively active RAC1 forms a complex with dynein in the CA RAC1 retina. (A) Retinal sections of CA RAC1 mice at P11 were immunostained with anti-rhodopsin antibody (gray or red). Nuclei were labeled with DAPI (blue). Rhodopsin-stained and merged images are shown. Mislocalized cells with nuclei in the INL extended long processes toward the ONL (indicated by arrows). Scale bar: 20 μm. (B) Retinal extracts (Extracts) of CA RAC1 mice at P11 were immunoprecipitated using either anti-RAC1 (RAC1-IP) or anti-MYC-tag (MYC-IP) antibody and control IgG (IgG-IP). Dynein intermediate chain (DIC), not kinesin heavy chain (KHC), was detected in both RAC1-IP and MYC-IP immunocomplexes by Western blotting. (C) Retinal sections of CA RAC1 mice at P11 were double immunostained with antibodies against MYC-Tag (green, CA RAC1) and DIC (red). Individual and merged images are shown, indicating that CA RAC1 colocalized with DIC. Scale bar: 10 μm.

Article Snippet: Examination was done on three to four animals for each genotype per age group (P6, P11, or P24). table ft1 table-wrap mode="anchored" t5 caption a7 Name Target Host Dilution Product Source MYC-Tag CA RAC1 Mouse 1:2,000 2276; Cell Signaling Technology, Inc., Danvers, MA, USA Rabbit 1:400 2272; Cell Signaling Technology, Inc. Cone arrestin Cone Rabbit 1:10,000 AB15282; Millipore, Temecula, CA, USA Rhodopsin Rod Mouse 1:10,000 O 4886; Sigma-Aldrich, St. Louis, MO, USA Retinoschisin IS marker Rabbit 1:1,000 Takada et al. 57 Synaptophysin Presynaptic marker Mouse 1:200 S 5768; Sigma-Aldrich PKCα Rod bipolar cell Rabbit 1:1,000 P 4334; Sigma-Aldrich PH3 Mitosis marker Rabbit 1:100 06-570; Millipore Ki67 Progenitor cell marker Mouse 1:400 556003; BD Biosciences, San Jose, CA, USA BrdU DNA synthesis marker Rat 1:100 Ab6326; Abcam, Inc., Cambridge, MA, USA PAR6 Polarity protein Rabbit 1:100 Sc-67393; Santa Cruz Biotechnology, Dallas, TX, USA PAR3 Polarity protein Rabbit 1:50 11085-1-AP; Proteintech Group, Chicago, IL, USA aPKCλ Polarity protein Rabbit 1:100 Sc-11399; Santa Cruz Biotechnology β-catenin OLM marker Mouse 1:300 610153; BD Biosciences DIC Dynein intermediate chain Mouse 1:400 MAB1618; Millipore Open in a separate window List of Primary Antibodies Used in Immunohistochemistry

Techniques: Labeling, Staining, Immunoprecipitation, Control, Western Blot