Co-IP and Western Blotting of HA-Tagged Proteins

Co-immunoprecipitation and western blotting assays were performed as previously described [20 (link), 32 (link)]. In brief, human 293T cells were lysed with the lysis buffer (150 mM NaCl, 50 mM Tris–HCl [pH 7.5], 1 mM EDTA, 1% Triton X-100, 0.5% NP-40, plus PMSF and protease inhibitor cocktail [Sigma]) for 30 min at 4 °C. The cell lysates were clarified by centrifugation at 18,000g for 30 min at 4 °C, then mixed with anti-HA agarose beads (Sigma) and incubated at 4 °C for 4 h, followed by washing four times with cold lysis buffer and eluting in gel loading buffer. As indicated, the beads were treated with RNase mixture (DNase-free, Roche) (20 μg/ml) and incubated at 37 °C for 30 min. The immunoprecipitated samples were analyzed by SDS-PAGE and detected by western blotting. Anti-HA antibody (mouse monoclonal, Covance), anti-FLAG antibody (rabbit polyclonal, MBL), anti-GAPDH antibody (rabbit polyclonal, MBL), anti-MOV10 antibody (rabbit polyclonal, Abcam), anti-ElonginC antibody (rabbit polyclonal, Abcam), anti-Cullin 5 antibody (rabbit polyclonal, Abcam), anti-A3G antibody (rabbit polyclonal, Abcam), anti-Vif antibody (mouse monoclonal, Abcam), and anti-HIV-1 p24 antibody (rabbitpolyclonal antibodies made by our lab) were used as primary antibodies [64 (link)]. Quantity One program (Bio-rad) was used to quantify the western blotting results.