Fig. 2

USP21 controls BRCA2 protein levels and ubiquitination. a Western blot analysis of the indicated proteins in the presence or absence of USP21 knockdown. Asynchronous U2OS cells were either left untreated or treated with CPT for 1 h. One of three representative experiments is shown. b BRCA2 and RAD51 protein turnover in HEK293T cells in the presence of USP21 knockdown or sh-Luc control. Western blot analysis was performed at the indicated timepoints after cycloheximide (CHX) treatment. A quantification of BRCA2 and RAD51 protein levels normalized to β-tubulin and 0 h CHX is shown. Values are expressed as mean and s.e.m. from three (2 h) or six independent experiments (4 and 8 h timepoints), p-values are based on Student’s two-tailed t-test: *p < 0.05, **p < 0.01. c Western blot analysis of BRCA2 and RAD51 levels in S phase cells. Following double-thymidine-block, cells were released for the indicated time frames prior to analysis. d IP using anti-HA conjugated beads in HEK293T cells transiently expressing empty vector or HA-USP21 in the presence or absence of CPT. Representative immunoblots from one of three independent experiments are shown. e Poly-ubiquitination analyses of the indicated proteins in cells expressing empty vector, Myc-USP21 WT (U21 WT) or Myc-USP21 C221A (U21 C221A). Endogenous poly-ubiquitin-chains were immunoprecipitated in the presence of MG-132 using TUBE1. Representative immunoblots from one of two independent experiments are shown