Fig. 4.

VHL modulates SARS-CoV nsp16 ubiquitination. (A) Left panels, HEK-293T cells were transfected with Myc-ubiquitin, Flag-nsp16 and an increasing amount of HA-VHL (1.0 and 2.0 μg). After 36 h transfection, the cells were collected and denatured in 1% SDS lysis buffer, and the cell lysate were diluted with lysis buffer until the concentration of SDS decreased to 0.1%. The cell extracts were used for co-immunoprecipitation with an anti-Flag antibody. The immunoprecipitates were detected by an anti-Myc or anti-Flag antibody. Right panels, assays were performed as in the left panels. SARS-CoV nsp14 as a negative control and Dvl2 as a positive control. (B) The HA-tagged ubiquitin mutants, Flag-nsp16 and Myc-VHL were transfected into HEK-293T cells. After 36 h, re-IP assays were performed with anti-Flag antibody. The immunoprecipitates were analyzed with an anti-HA or anti-Flag antibody. (C) The HA-ubiquitin, Flag-nsp16 or Flag-nsp14 or Flag-Dvl2, control (PLKO.1) or VHL RNAi plasmids (1# or 2#) were transfected into HEK-293T cells. After 24 h, the cells were treated with puromycin (1 μg/ml) for another 24 h and then lysed for re-IP assays with anti-Flag antibody. The immunoprecipitates were analysed with the anti-HA or anti-Flag antibody (upper panels).