(A) The protein level of HRD1, S100A8 through Western blotting analysis in MCF7 cells with overexpression of different dose of HRD1. (B) The protein level of HRD1 in MCF7 cells by knockdown HRD1 through Western blotting analysis. (C) MCF7 cells were transfected with si-HRD1 and the control for 48 h, followed by exposure to cycloheximide (CHX 50ng/ml) for 0, 2, 4, 6 h. The protein of S100A8 and HRD1 in whole cell lysates was measured. (D) Ubiquitination of S100A8 was induced by HRD1. HA-ubiquitin was co-expressed in MCF7 cells with His-HRD1 or Vector control with treatment of MG132 (10 μmol/l) for 4 h. Ubiquitinated S100A8 was immunoprecipitated using S100A8 antibody and further detected with UB antibody. The endogenous S100A8 and His-HRD1 in the whole cell lysates were examined by S100A8 and His-tag antibodies. *P < 0.05, compared to Si-NC.