Methylation of the Tumor Suppressor Protein, BRCA1, Influences Its Transcriptional Cofactor Function
Figure 1
BRCA1 is methylated at both arginine and lysine residues in breast cancer cell lines.
(a) Predicted BRCA1 methylation sites generated by Memo: Methylation Modification Prediction Server (http://www.bioinfo.tsinghua.edu.cn/~tigerchen/memo.html) R = Arginine and K = Lysine. (b) Two milligram of whole cell protein extracts from MCF-7 and MDA-MB-231 cells were immunoprecipitated with either BRCA1(C-20) or rabbit normal IgG antibody, separated on a 4–20% gel by SDS-PAGE, and western blotted with antibodies against K-methyl, R-methyl and BRCA1(C-20). Input represents 1/10 of immunoprecipitated material. Results are representative of three independent experiments. (c) One milligram of whole cell protein extracts from MDA-MB-231 and BRCA1-null UWB1.289 cells were immunoprecipitated with either BRCA1(C-20) or rabbit normal IgG antibody, separated on a 4–20% gel by SDS-PAGE, and western blotted with antibodies against R-methyl and BRCA1(C-20) in order to validate observed methylated bands. Densitometry of R-methylation was normalized to amount of detected immunoprecpipitated BRCA1, background subtracted based on IgG counts. (d) One milligram of whole cell protein extracts from synchronized MDA-MB-231 cells were immunoprecipitated with either BRCA1(C-20) or rabbit normal IgG antibody, separated on a 4–20% gel by SDS-PAGE, and western blotted with antibodies against R-methyl and BRCA1(C-20). Densitometry of R-methylation was normalized to amount of detected immunoprecpipitated BRCA1, background subtracted based on IgG counts. Input represents 1/10 of immunoprecipitated material. (e) Whole cell protein extracts from synchronized MDA-MB-231 cells were separated on a 4–20% gel by SDS-PAGE, and western blotted with antibodies against Cyclin B(H-433), Cyclin D1(M-20), Cyclin E(C-19), cdk4(H-303) and actin(C-11).