Fig. 5.

RING domain of BRCA1 is not important for CTSS-mediated BRCA1 degradation. a Western blot analysis in WT-BRCA1 and RING domain deleted mutant (ΔRING) transfected MCF7 cells treated with 100 μg/ml cycloheximide (CHX) for various lengths of time. b MCF7 cells were transfected with WT-BRCA1 or ΔRING with or without siRNA of CTSS (si-CTSS) and incubated in the presence of CHX and Western blotting was performed. Band density was expressed as the fold change relative to the control (Mean ± SD of 3 experiments). c MCF7 cells were transfected with WT-BRCA1, ΔRING, or ΔBRCT. For ubiquitination assays, MG132 (10 μM) was treated after transfection of WT-CTSS, and cell lysates were immunoprecipitated (IP) and immunoblotted (IB) with ubiqutin construct (Ub). d Promoter reporter construct gadd45 in MCF7 (left) and MDA-MB-231 (right) cells were co-transfected with WT-BRCA1, ΔRING, or ΔBRCT as indicated. Normalized luciferase activity referred to the activity of the extracts. Data are representative of three independent experiments with similar results. Graphs represent mean ± SD of three experiments. *p < 0.05 (ANOVA). e Cell lysates after transfection of sh-CTSS or treatment of VBY-036, a CTSS specific inhibitor at 10 μM were immunoprecipitated (IP) with cyclin B1 and immunoblotted (IB) with ubiqutin construct (Ub). Western blotting was also performed