Supplementary Figure 6: Loss of CHK2 or loss the CHK2-mediated phosphorylation of BRCA1 causes enhanced interaction of BRCA1 with active Aurora-A.

(a) Representative western blots showing the re-expression of wild type (WT), non-phosphorylatable (S988A) and phospho-mimetic (S988E) mutants of BRCA1 in HCT116 cells stably expressing shRNAs targeting endogenous BRCA1. Relative protein levels were quantified. (b) The interaction of BRCA1 with active Aurora-A is increased when CHK2 is decreased. Immunoprecipitation of BRCA1 from whole cell lysates derived from HCT116 cells with or without low levels of CHK2 (mediated by stable expression of shRNAs targeting CHK2) or after reconstitution of CHK2 expression (mediated by stable expression of shRNA resistant mutant of CHK2). Cells were synchronized in mitosis, BRCA1 was immunoprecipitated and associated Aurora-A and active Aurora-A proteins (auto-phosphorylated at threonine-288; P-Aurora-A) were subsequently detected on western blots. Active Aurora-A bound to BRCA1 is significantly enhanced when CHK2 expression is repressed. Representative western blots are shown. (c) The interaction of BRCA1 with active Aurora-A is increased after loss of the CHK2 phosphorylation site of BRCA1. Immunoprecipitation of BRCA1 from mitotic synchronized, stable HCT116 + BRCA1 shRNA cells expressing either a wild type (WT), a non-phosphorylatable mutant (S988A) or a phospho-mimetic mutant (S988E) of BRCA1 and subsequent detection of active Aurora-A (P-Aurora-A, P-Thr288). BRCA1, which cannot be phosphorylated by CHK2 shows an enhanced interaction with the active form of Aurora-A. Representative western blots are shown. (d) Control immunoprecipitation experiments showing that the interaction of Aurora-A with its co-factor TPX2 is not altered in the absence of CHK2. Immunoprecipitation of TPX2 from CHK2 proficient (HCT116) or deficient (HCT116-CHK2^−/−) mitotic synchronized cells and subsequent detection of Aurora-A on western blots. Representative western blots are shown.