Figure 5. Expression and stability of Vpr natural variants.

(a) 293 T cells were transfected with myc fusion constructs of wild type B Vpr, C Vpr and Vpr variants (2 µg each) using Lipofectamine 2000; the cell lysates were prepared 24 hrs post-transfection and were run on 12 percent SDS PAGE. Western blotting was done using c-myc monoclonal mouse antibody as primary antibody and HRP labeled anti-mouse antibody as secondary antibody. GAPDH was probed as a loading control. (b) 293T cells were transfected with 2 µg each of myc fusion clones of wild type subtype B Vpr, C Vpr and Vpr variants and after 24 hrs of transfection, cells were treated with cycloheximide, CHX (100 µg/ml) and harvested at the intervals of 2 hrs upto 6 hrs. Cell lysates were resolved by 12 percent SDS PAGE and blots were probed with c-myc monoclonal antibody and GAPDH antibody (loading control) (c) The relative concentration of protein at different time points was measured was measuring the density of the band in the each case and were line plotted against the duration of CHX treatment.