Research Paper Volume 3, Issue 5 pp 515—532
Mutations in the BRCT binding site of BRCA1 result in hyper-recombination
Figure 4. K1702M increases ubiquitination of the BASC which is required for hyper-recombination. (A) HCC1937/DR-GFP cells infected with the indicated HD-Ad vectors were analyzed for HRR/GFP events by FACS. Error bars show SEM from three independent experiments (***, P < 0.001; ns, not significant). P < 0.0001 when K1702M was compared to I26A/K1702M. P = 0.1780 when wild-type BRCA1 was compared to I26A. (B) Western blot analysis for FLAG-BRCA1 expression in infected HCC1937 cells with DNA-PKcs as loading control. (C) HEK293T cells were co-transfected with Myc-ubiquitin plasmid and the indicated FLAG-BRCA1 plasmids. Immuno-precipitated FLAG material was examined for ubiquitin by western blot analysis. The smear in each of the lanes represents the many proteins in the BASC that are ubiquitinated. (D) The indicated FLAG-BRCA1 plasmids were co-transfected with Myc-CtIP plasmid and immuno-precipitated FLAG material was examined for CtIP binding by western blot analysis.