Figure 3

Effect of Hrd1 on CDV H protein level. A Overexpression of Hrd1-negatively regulated CDV H protein level. 293 T cells were transfected with Flag-CDV H alone or together with Myc-Hrd1 for 24 h, the protein levels were determined 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. B Increased CDV H protein level by knockdown of Hrd1. 293 T cells were transfected with siRNA for Hrd1 for 24 h and then transfected with Flag-CDV H for an additional 24 h. The indicated proteins were visualized by Western blot using anti-Flag, anti-Hrd1, 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. C Hrd1 degradation of CDV H protein through proteasome pathway. 293 T cells were transfected with Flag-CDV H alone or together with Ha-Hrd1 for 24 h and then treated with or without MG132 for 6 h. Protein levels were assessed by Western blot using anti-Flag, anti-Ha and anti-GAPDH antibodies. The level of CDV H protein was quantified by determining band intensities and subsequent normalization to the level of GAPDH. All results are shown as means ± SD from at least three separate sample preparations. * p < 0.05, ** p < 0.01.