Figure 4

Critical role of E3 ligase activity on Hrd1-mediated CDV H protein degradation. A Schematic of the RING domain of Hrd1. The red C329S mutation in RING domain could abolish the E3 ligase activity of Hrd1. The partial DNA sequence that contains normal WT and mutant Hrd1 were aligned and the conserved cysteine residue in position 329 of the RING domain was replaced by a serine residue. B Hrd1 with C329S mutation did not degrade CDV H. 293 T cells were transfected with Flag-CDV H alone or together with Myc-Hrd1 or C329S mutant for 24 h, and proteins were analyzed by Western blot using anti-Flag, anti-Myc and anti-GAPDH antibodies. The level of CDV H protein was quantified by determining band intensities, which were then normalized to the level of GAPDH. Data are means ± SD (*p < 0.05). C Hrd1 with C329S mutation did not modify ubiquitination of CDV H protein. Ha-Ub and Flag-CDV H were co-transfected into 293 T cells together with Myc-Hrd1 or Myc-C329S mutant for 24 h. The cells were lysed and immunoprecipitated with anti-Flag antibody. The input samples and co-precipitated proteins were analyzed by Western blot with anti-Ha, anti-Flag, anti-Myc and anti-GAPDH antibodies. The ubiquitination level of CDV H protein was quantified by determining band intensities, which were then normalized to immunoprecipitated CDV H protein. Data are means ± SD (*p < 0.05).