Figure 2.

USP9X knockdown reduces BRCA1 stability and enhances its ubiquitination. A, Lysates from cells stably expressing shNC, shUSP9X#1 and shUSP9X#2 were subjected to immunoblotting analysis with the indicated antibodies. B and C, MCF‐7 and HeLa cells stably expressing shNC or shUSP9X were treated with 200 μg/mL cycloheximide (CHX) for the indicated times. Total cellular lysates were subjected to immunoblotting analysis with the indicated antibodies (B). Quantitative results of relative BRCA1 protein levels (BRCA1/Vinculin) from three independent experiments are shown in C. D, HEK293T cells were cotransfected with Flag‐BRCA1, HA‐ubiquitin (Ub), siNC, or siUSP9Xs (#1‐3) for 48 h. Then, cells were treated with 20 μmol/L MG‐132 for 6 h and then subjected to immunoprecipitation assays using Flag M2 affinity gel, followed by immunoblotting analysis with the indicated antibodies