rabbit polyclonal anti e2f4  (Bethyl)

Journal: Cancer Cell International

Article Title: Long non-coding RNA GAS5 accelerates oxidative stress in melanoma cells by rescuing EZH2-mediated CDKN1C downregulation

doi: 10.1186/s12935-020-01167-1

Figure Lengend Snippet: Overexpression of GAS5 prevents H3K27 trimethylation and upregulates CDKN1C expression to accelerate oxidative stress in melanoma by inhibiting EZH2. a Correlation analysis of lncRNA GAS5 expression and EZH2 expression. b RIP assay of the recruitment of transcription factor E2F4 by lncRNA GAS5. c ChIP assay of the enrichment of E2F4 in EZH2 promoter region. d RNA pull-down assay of the direct interaction of lncRNA GAS5 and E2F4. A375 cells were treated with oe-GAS5 alone or in the presence of oe-EZH2. e RT-qPCR assay of lncRNA GAS5 expression and mRNA expression of EZH2 and CDKN1C. f Western blot assay of the protein expression of E2F4, EZH2, H3K27me3 and CDKN1C. g Western blot assay of the protein expression of MDA5, IRE1α and SOD-1. h ELISA analysis of the content of ROS. i CCK-8 assay of cell viability. j Flow cytometric analysis of cell apoptosis. * p < 0.05, compared with the IgG group, cells transduced with oe-NC, Bio-probe NC group, cells transduced with oe-G-NC or cells transduced with oe-GAS5 + oe-E-NC. The above measurement data are expressed as mean ± standard deviation. The unpaired t -test is used for comparison of the data between two groups. Data among multiple groups are analyzed by one-way ANOVA, followed by Tukey’s post hoc test. The statistical analysis concerning time-based measurements within each group is realized using ANOVA of repeated measurements, followed by a Bonferroni’s post hoc test. Pearson correlation analysis is performed for correlation analysis

Article Snippet: The antibodies were recruited for RIP including the primary rabbit polyclonal antibody to E2F4 (ab245449, 1: 50, Abcam Inc., Cambridge, UK) and the rabbit anti-human IgG (ab109489, 1: 100, Abcam Inc., Cambridge, UK) as an NC.

Techniques: Over Expression, Expressing, Pull Down Assay, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, CCK-8 Assay, Transduction, Standard Deviation, Comparison

Journal: Nature communications

Article Title: ZC3H18 specifically binds and activates the BRCA1 promoter to facilitate homologous recombination in ovarian cancer.

doi: 10.1038/s41467-019-12610-x

Figure Lengend Snippet: Fig. 6 ZC3H18 binds the BRCA1 promoter and inhibits E2F1 binding. a Schematic of the BRCA1 proximal promoter with E2FA and E2FB sites indicated. Nucleotide sequences of the DNA probes used in the electrophoretic mobility shift assays (EMSA). E2FA and E2FB mutation sites are indicated in open rectangles. b EMSA with purified recombinant SFB-ZC3H18 using BRCA1 promoter probe with wild-type sequence (E2FA/BWT) or mutations in the E2FA site (E2FΔA), the E2FB site (E2FΔB), or both E2F sites (E2FΔA/B). A probe with randomly shuffled sequences was used as negative control. For supershift assays, an anti-S-Tag monoclonal antibody, which binds the SFB tag in SFB-ZC3H18, was used. c, d ZC3H18 and E2F4 co-occupy the endogenous BRCA1 promoter. Sequential ChiP (ChIP-Re-ChIP) assays in OVCAR-8 cells using anti-ZC3H18 antibody for primary ChIP and anti-E2F4 antibody for secondary ChIP (c) and using anti-E2F4 antibody for primary ChIP and anti-ZC3H18 antibody for secondary ChIP (d). e EMSA with purified recombinant SFB-ZC3H18 and SFB-E2F1 using BRCA1 promoter probe with mutated E2FB site (E2FΔB). f EMSA with purified SFB-E2F4 and SFB-E2F1 using BRCA1 promoter probe with mutated E2FA site (E2FΔA). The images of EMSA in b, e, and f are representative of three independent experiments that gave similar results. Data in c and d are means ± SEM of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, unpaired Student’s t-test

Article Snippet: Immunoblotting was done using the following primary antibodies: rabbit polyclonal ZC3H18 (1:1000, A304-682A, Bethyl Laboratories Inc.); mouse monoclonal BRCA1 (1:2000, sc-6954, Santa Cruz Biotechnology); mouse monoclonal E2F1 (1:500, ab4070, Abcam); rabbit polyclonal E2F4 (1:1000, NBP1-21374, Novus Biologicals); mouse monoclonal DNMT1 (1:5000, ab13537, Abcam); mouse monoclonal HSP90 (D. Toft, Mayo Clinic, H9010), and rabbit monoclonal HA-tag (1:1000, CST-3724S, Cell Signaling Technology).

Techniques: Binding Assay, Electrophoretic Mobility Shift Assay, Mutagenesis, Recombinant, Sequencing, Negative Control, ChIP-chip

Journal: Nature communications

Article Title: ZC3H18 specifically binds and activates the BRCA1 promoter to facilitate homologous recombination in ovarian cancer.

doi: 10.1038/s41467-019-12610-x

Figure Lengend Snippet: Fig. 7 ZC3H18 and E2F4 expression correlates with BRCA1 levels in HGSOC patient and PDX tumors. a Scatter plots of BRCA1 mRNA expression as a function of either ZC3H18 or E2F4 mRNA expression in HGSOC tumors from patients and PDX models. mRNA expression is in RPKM units. b Model for the role of ZC3H18 in BRCA1 transcription. Left panel: in ZC3H18-proficient cells, ZC3H18 directly binds to the E2FA site on the BRCA1 promoter, where it promotes E2F4 occupancy at the E2FB site, thereby preventing E2F1-dependent DNMT1 occupancy and promoter methylation and inducing BRCA1 transcription. Right panel: in ZC3H18-deficient cells, E2F1 occupies both E2FA and E2FB sites and causes DNMT1 loading onto the promoter, leading to methylation of the promoter, reduced expression of BRCA1, and disruption of HR. Spearman correlations are shown in the images

Article Snippet: Immunoblotting was done using the following primary antibodies: rabbit polyclonal ZC3H18 (1:1000, A304-682A, Bethyl Laboratories Inc.); mouse monoclonal BRCA1 (1:2000, sc-6954, Santa Cruz Biotechnology); mouse monoclonal E2F1 (1:500, ab4070, Abcam); rabbit polyclonal E2F4 (1:1000, NBP1-21374, Novus Biologicals); mouse monoclonal DNMT1 (1:5000, ab13537, Abcam); mouse monoclonal HSP90 (D. Toft, Mayo Clinic, H9010), and rabbit monoclonal HA-tag (1:1000, CST-3724S, Cell Signaling Technology).

Techniques: Expressing, Methylation, Disruption