Journal: eLife

Article Title: Distinct and diverse chromatin proteomes of ageing mouse organs reveal protein signatures that correlate with physiological functions

doi: 10.7554/eLife.73524

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-RNF20 (rabbit polyclonal) , Cell Signaling Technology , Cat# 9425S;RRID: AB_2797700 , WB (1:1000).

Techniques: Software

Journal: eLife

Article Title: Distinct and diverse chromatin proteomes of ageing mouse organs reveal protein signatures that correlate with physiological functions

doi: 10.7554/eLife.73524

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-RNF20 (rabbit polyclonal) , Cell Signaling Technology , Cat# 9425S;RRID: AB_2797700 , WB (1:1000).

Techniques: Software

Journal: Nucleic Acids Research

Article Title: Bimodal regulation of the PRC2 complex by USP7 underlies tumorigenesis

doi: 10.1093/nar/gkab209

Figure Lengend Snippet: The assembly of the USP7/EZH2 complex on transcriptional targets. ( A and B ) qChIP analysis of selected promoters in the A375 cells after co-transfection with indicated shRNA and Vector or FLAG-EZH2 using the indicated antibodies. The knockdown efficiencies of USP7 and EZH2 were verified by western blotting. ( C ) Western blotting analysis of the expression of indicated proteins in A375 cells that transfected with different sets of USP7 siRNAs. ( D ) Western blotting analysis of the expression of H3K27me3 and H2AK119ub in A375 cells transfected with the indicated siRNAs. ( E ) A375 cells were transfected with indicated siRNAs for qChIP analysis on the selected promoters using antibodies against the indicated histone modification. ( F ) A375 cells transfected with FLAG-RNF20 or FLAG-RNF40 for the measurement of the indicated histone modification by western blotting. ( G ) A375 cells were transfected with the indicated expression vectors for qChIP analysis on the selected promoters using antibodies against the indicated histone modification. In A, B, E and G, data represent the mean ± SD from biological triplicate experiments. * P < 0.05 and ** P < 0.01, one-way ANOVA.

Article Snippet: The sources of antibodies against the following proteins were as follows: HA (sc-805) from Santa Cruz Biotechnology; PCGF1 (PA5-49390, for WB), β-actin (A1978), EZH2 (AV38470, for IHC), RYBP (PRS2227, for WB) and FLAG (F3165) from Sigma; USP7 (05-1946, for WB, IF and IP) and H2AK119ub1 (ABE569, for WB and ChIP) from Millipore; Ubiquityl-Histone H2B (K120) (#5546, for WB, IF and ChIP), SKP1 (#2156, for WB), Histone H2A (#2578, for WB and ChIP), RNF20 (#11974, for WB), RNF40 (#12187, for WB) and FOXO1 (#2880, for WB and IHC) from Cell Signaling Technology; KDM2B (ab234082, for WB), SUZ12 (ab175187, for WB and IP), H3K27me3 (ab6002, for WB, IF, ChIP, and ChIP-seq), H2B (ab64165, for WB and ChIP), DDDDK-tag (ab1257, for IF) and histone H3 (ab1791, for WB and ChIP) from Abcam; BCOR (12107-1-AP, for WB) from Proteintech; USP7 (A300-033A, for IP, IF, IHC and ChIP), RING1A (A303-552A, for WB) and RING1B (A302-869A, for WB) from Bethyl Lab; Myc (M047-3) from MBL; EED (GTX33168, for WB and IP) and YAF2 (GTX115355, for WB) from GeneTex; EZH2 (612667, for WB, IP and IF) from BD Transduction Laboratories; and EZH2 (39901, for ChIP and ChIP-seq) from Active Motif.

Techniques: Cotransfection, shRNA, Plasmid Preparation, Western Blot, Expressing, Transfection, Modification

Journal: Frontiers in Oncology

Article Title: RNF20 Is Critical for Snail-Mediated E-Cadherin Repression in Human Breast Cancer

doi: 10.3389/fonc.2020.613470

Figure Lengend Snippet: RNF20 alters the expression of epithelial-mesenchymal transition (EMT) markers. (A) Expression of E-cadherin, Vimentin and RNF20 in the MDA-MB231 and BT549 cells was analyzed by Western blotting. Actin was served as a loading control. (B) Expression of RNF20 and E-cadherin was analyzed by qPCR in MDA-MB231 and BT549 cells infected with empty vector (Ctr) or RNF20-expressing vector. *P < 0.01 by Student’s t test. (C) Expression of E-cadherin, and RNF20 was analyzed by Western blotting in BT474 cells with stable empty vector or knockdown of RNF20 expression as well as shRNF20-expressing BT474 cells infected with plvx-neo-RNF20. (D) Expression of RNF20 and E-cadherin was analyzed by qPCR in cells from (C) . *P < 0.01 by Student’s t test.

Article Snippet: RNF20 antibodies and Snail antibodies were from Abcam and Cell Signaling Technology respectively.

Techniques: Expressing, Western Blot, Infection, Plasmid Preparation

Journal: Frontiers in Oncology

Article Title: RNF20 Is Critical for Snail-Mediated E-Cadherin Repression in Human Breast Cancer

doi: 10.3389/fonc.2020.613470

Figure Lengend Snippet: RNF20 enhances breast cancer cell migration and invasion in vitro . (A–C) Migratory (A, B) and invasive ability (C) of MDA-MB231 and BT549 cells with stable empty vector or RNF20 expression were analyzed. The percentage of migratory and invasive cells is shown in the bar graphs (mean ± SD in three separate experiments). *P < 0.01 by Student’s t test: 100 µm.

Article Snippet: RNF20 antibodies and Snail antibodies were from Abcam and Cell Signaling Technology respectively.

Techniques: Migration, In Vitro, Plasmid Preparation, Expressing

Journal: Frontiers in Oncology

Article Title: RNF20 Is Critical for Snail-Mediated E-Cadherin Repression in Human Breast Cancer

doi: 10.3389/fonc.2020.613470

Figure Lengend Snippet: RNF20 interacts with Snail. (A) 293T cells were transiently coexpressed with Flag-tagged RNF20 and HA-tagged Snail. Cell extracts were immunoprecipitated separately with Flag or HA antibodies, and the associated RNF20 and Snail were examined by Western blotting, respectively. (B) Schematic diagram showing the structure of Snail and two deletion mutations. (C) Full-length and deletion mutants of Snail were coexpressed with RNF20 in 293T cells. After immunoprecipitation of RN20, associated Snail was analyzed by Western blotting.

Article Snippet: RNF20 antibodies and Snail antibodies were from Abcam and Cell Signaling Technology respectively.

Techniques: Immunoprecipitation, Western Blot

Journal: Frontiers in Oncology

Article Title: RNF20 Is Critical for Snail-Mediated E-Cadherin Repression in Human Breast Cancer

doi: 10.3389/fonc.2020.613470

Figure Lengend Snippet: RNF20 interacts with G9a. 293T cells were transiently coexpressed with Flag-tagged G9a, HA-tagged RNF20. Cell extract were immunoprecipitated separately with Flag or HA antibodies, and the associated RNF20 and G9a were examined by Western blotting respectively.

Article Snippet: RNF20 antibodies and Snail antibodies were from Abcam and Cell Signaling Technology respectively.

Techniques: Immunoprecipitation, Western Blot

Journal: Frontiers in Oncology

Article Title: RNF20 Is Critical for Snail-Mediated E-Cadherin Repression in Human Breast Cancer

doi: 10.3389/fonc.2020.613470

Figure Lengend Snippet: RNF20 is recruited to the E-cadherin promoter for epigenetic silencing of E-cadherin expression by Snail. (A, B) RNF20 was expressed in MDA-MB231 and BT549 cells, and RNF20, H3K9me2, and H3K9 acetylation at the E-cadherin promoter were analyzed by the ChIP assay. Quantitative real-time PCR was also performed to analyze ChIP samples in (A) (mean ± SD from three separate experiments). *P < 0.01 by Student’s t test.

Article Snippet: RNF20 antibodies and Snail antibodies were from Abcam and Cell Signaling Technology respectively.

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: Frontiers in Oncology

Article Title: RNF20 Is Critical for Snail-Mediated E-Cadherin Repression in Human Breast Cancer

doi: 10.3389/fonc.2020.613470

Figure Lengend Snippet: RNF20 promotes tumorsphere and colony formation. (A) Tumorsphere formation of MDA-MB231 and BT549 cells with stable empty vector (Ctr) or overexpression of RNF20 was measured. (B) The formation of colonies was measured from MDA-MB231 and BT549 cells with stable empty vector (Ctr) or overexpression of RNF20, as well as BT474 cells with stable empty vector (Ctr) or knockdown of RNF20 expression. Data are presented as a percentage of empty vector cells lines (mean ± SD in three separate experiments). *P < 0.01 by Student’s t test (C) Kaplan-Meier survival analysis for overall survival (OS) of patients in a breast cancer dataset according to RNF20 protein expression status. The p value was determined using the log-rank test.

Article Snippet: RNF20 antibodies and Snail antibodies were from Abcam and Cell Signaling Technology respectively.

Techniques: Plasmid Preparation, Over Expression, Expressing

Journal: Nucleic Acids Research

Article Title: Egr2-guided histone H2B monoubiquitination is required for peripheral nervous system myelination

doi: 10.1093/nar/gkaa606

Figure Lengend Snippet: Physical and functional interaction of Egr2 and Rnf40/Rnf20. ( A ) Venn diagram depicting the overlap of Egr2 target genes with lost H2Bub1 marks in Rnf40 ΔSC nerves. ( B , C ) GO analysis (B) and list (C) of Egr2 target genes with reduced H2Bub1 marks in Rnf40 ΔSC nerves. ( D ) Co-immunoprecipitation (IP) of Rnf40 and Rnf20 with antibodies directed against Egr2 and pre-immune serum (PI) from extracts of HEK293T cells that were transfected with various expression plasmids as indicated below the panels. Western blot was used to detect precipitated Rnf40 (upper panel), Rnf20 (middle panel) and Egr2 (lower panel). Numbers on the right indicate position of co-electrophoresed size markers in kDa. ( E ) Scheme of Egr2 proteins and their ability to interact with myc-tagged Rnf40 in extracts from transfected HEK293T cells. DUF3446, domain of unknown function. ( F ) Scheme of myc-tagged Rnf40 proteins and their ability to interact with Egr2 in extracts from transfected HEK293T cells. ( G ) Scheme of HA-tagged Rnf20 proteins and their ability to interact with Egr2 in extracts from transfected HEK293T cells. ( H , I ) Co-immunoprecipitation (IP) of Egr2 with antibodies directed against Rnf40 (H), Rnf20 (I) and IgG control (H, I) from extracts of differentiating SC cultures. Western blot was used to detect precipitated Egr2 (upper panels), Rnf40 (lower panel, H) and Rnf20 (lower panel, I). Experiments were repeated three times. Uncropped western blots for D–I are presented as . ( J – M ) Determination of endogenous expression levels of Mbp (J), Mpz (K), Mag (L) and Prx (M) by qRT-PCR in Neuro2a cells transfected with various combinations of expression plasmids for Egr2, Rnf40-specific shRNA (shRnf40), scrambled shRNA (shScr) or various combinations of these as indicated below the lanes after FACS. Transcript levels in cells transfected with empty expression vectors (ctrl) were set to 1 and all other values were expressed in relation to it ( n = 3; mean values ± SEM). Statistical significance was determined by one-way ANOVA with Bonferroni's multiple comparisons test (* P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001). Exact values are provided in the Supplementary Tables. ( N ) Proposed model for the action of the Rnf40/Rnf20 E3 ligase and H2Bub1 modification in SC development.

Article Snippet: For stainings, the following primary antibodies were used: chicken anti-Mpz antibodies (Aves Labs, #PZO, Lot #PZO8767965, 1:500 dilution), goat anti-Sox2 antiserum (Santa Cruz, #sc17319, Lot #I1115, 1:500 dilution), goat anti-Sox10 antiserum (home-made, generated against a bacterially expressed and purified peptide corresponding to amino acids 181–233 and 308–400 of rat Sox10 according to accession number NM_019193.2, validated on control and knockout tissue, 1:3000), guinea pig anti-Sox10 antiserum (home-made, validated on control and knockout mouse tissue, 1:1000 dilution) , guinea pig anti-Egr2 antiserum (home-made, validated on mouse sciatic nerve, 1:1000 dilution) , rabbit anti-Egr2 antiserum (Covance, #PRB-236P, Lot #D13BF00486, 1:200 dilution), rabbit anti-Rnf40 monoclonal (Abcam, #ab191309, Lot #11974, 1:100 dilution), rabbit anti-Rnf20 monoclonal (Cell signaling, #11974, Lot #1, 1:100 dilution), rabbit anti-Oct6 antiserum (home-made, validated on control and knockout mouse tissue, 1:2000 dilution) , rabbit anti-Nav1.6 antiserum (Alomone Labs, #ASC-009, Lot#ASC009AN2425, 1:50 dilution), rabbit anti-Caspr antiserum (Abcam, #ab34151, Lot #GR86230, 1:1000 dilution), rabbit anti-Iba1 antiserum (Wako, #019–19741, Lot #SAE6921, 1:250 dilution), rabbit anti-Ki67 antiserum (Thermo Fisher Scientific, #RM-9106, Lot#9106S906D, 1:500 dilution), rat anti-Ki67 antiserum (Thermo Fisher Scientific, #14–5698-82, Lot #2056928, 1:500 dilution), rat anti-MBP monoclonal (Bio-Rad, #MCA409S, Lot #210610, 1:300 dilution), mouse anti-H2Bub1 monoclonal (gift of D. Eick, Helmholtz Zentrum München, 1:5 dilution) ( ).

Techniques: Functional Assay, Immunoprecipitation, Transfection, Expressing, Western Blot, Quantitative RT-PCR, shRNA, Modification