Fig. 7: A protein complex containing p85β/BCLAF1/TRIM28/ZNF263 binds to and regulates BCLAF1 promoter.
From: p85β acts as a transcription cofactor and cooperates with BCLAF1 in the nucleus
A A schematic of the BCLAF1 gene showing the putative ZNF263 recognition motif within locus 1, locus 2 and promoter region. The mutated nucleotides within the motif in the mutant BCLAF1 promoter reporter are highlighted in red. B Cytoplasmic and nuclear extracts of OVCAR8 cells were subjected to immunoprecipitation (IP) with anti-p85β antibody, anti-ZNF263 antibody or anti-TRIM28 antibody. IP with IgG was control and input represents total cell lysate. C Lysates of SKOV3 stably expressing vector or PIK3R2 (R2-OX) were subjected to IP with anti-BCLAF1 antibody. BCLAF1 protein levels were normalized prior to IP by using proportional amounts of lysates. The numbers below the blots are densitometry values relative to vector control. The bar graphs display the mean relative densitometry values from three independent experiments. D SKOV3 cells were subjected to proximity ligation assay with anti-ZNF263 and anti-BCLAF1 antibodies. The individual antibody alone served as negative control. The number of signals per nucleus of 200 cells from three independent experiments is presented as mean ± SD. Scale bars, 9 μm. E, F Cross-linked chromatin from DOV13 cells stably expressing PIK3R2 was immunoprecipitated with (E) anti-TRIM28 antibody or (F) anti-ZNF263 antibody or rabbit IgG. DNA fragments were amplified with primers for BCLAF1 promoter. G–I re-ChIP was performed with the indicated sequence of antibodies, with the first antibody against (G) BCLAF1, (H) ZNF263 or (I) HA. Normal rabbit IgG in second ChIP served as control. J DOV13 or SKOV3 cells were transfected with human WT (pGL3-BCLAF1-luc) or mutant (pGL3-BCLAF1-mut-luc) BCLAF1 promoter luciferase reporter or pGL3-Basic plasmid for 48 h. K ChIP-PCR for ZNF263 at BCLAF1 promoter in SKOV3 cells transfected with PIK3R2 siRNA for 72 h. The blots in (B, C) are representative of three independent experiments. Each individual data point in (E–K) represents the average of triplicates from one independent experiment; therefore, the data shown (mean ± SD) are from three independent experiments. P-values were obtained using two-tailed t-test (C–I) or two-way ANOVA with Tukey’s post hoc test (J, K). Source data are provided as a Source Data file.
