Fig. 3.

VHL regulates SARS nsp16 protein stability, degradation, and SARS replication. (A) Flag-nsp16 and Flag-VHL plasmids were transfected into HEK293T cells. After 24 h, the cells were treated with CHX (100 μg/ml) for the different times. The cell lysates were analyzed by Western blotting with an anti-Flag or anti-β-actin antibody. (B) Flag-nsp16 and HA-VHL plasmids were transfected into HEK293T cells. The cells were treated with MG132 (20 μM) or NH₄Cl (10 mM) for another 6 h after 24 h transfection. The cell lysates were analyzed by Western blotting with different antibodies. (C) HEK293T cells were transfected with Flag-nsp16 and control (PLKO.1) or VHL knockdown plasmids (sh-VHL-1#, sh-VHL-2#). After 24 h, the cells were treated with puromycin (1 μg/ml) for an additional 16 h. The cell lysates were analyzed by Western blotting with indicated antibodies. (D) The SARS-rep-LUC (0.1 μg) and HA-VHL (0.3 μg) were transfected into Vero cells. The pcDNA3.0 and pRL-TK were used to make an equal amount of total DNA and normalize the transfection efficiency. The reporter assays were processed 36 h after transfection. (E) Assays were performed as in (D) except for the transfection with VHL RNAi plasmids. *, p < 0.05, **, p < 0.01; the bar graphs show the mean value ± SD of triplicate samples.