Investigating APOBEC3G Regulation by HIV-1 Vif

We followed a method in ref. ^{23 (link)}. Briefly, human embryonic kidney (HEK293T) cells in 12-well plates were co-transfected with pcDNA3.1-based A3G, sA3G, or their mutant expression vector (0.66 μg) and pcDNA-HVif, pcDNA-HVif-H41A/H42A or empty pcDNA3.1 vector (1.32 μg) with the transfection reagent, FuGene HD (Promega). After incubation for 24 h, 2 μM MG132 or DMSO was added to the medium, and cells were incubated for an additional 24 h. Resulting cell lysates were analyzed by SDS-PAGE and immunoprobed with monoclonal anti-c-myc (9E11, Abcam), anti-Vif (319, Abcam), or anti-α-tubulin primary antibodies (DM1A, Abcam), and monoclonal anti-mouse IgG secondary antibody conjugated with a fluorophore (ab216772, Abcam). Protein bands were detected with a ChemiDoc system (Bio-Rad Laboratories) and analyzed using NIH ImageJ^{59 (link)}.