Anti α tubulin mab from mouse

FLAG-A3H-pcDNA or HA-A3H-pcDNA variants were co-transfected with either Vif-pcDNA or pcDNA3.1(+) empty vector into HEK293T cells (ATCC) by using X-tremeGENE 9 DNA Transfection Reagent (Roche). At post-48 h transfection, the cells were washed once with PBS and lysed in RIPA buffer with 1× complete protease inhibitors (Roche). The lysates were then subjected to Western blot with anti-FLAG M2 mAb from mouse (Sigma-Aldrich, 1:3000 dilution), anti-HA mAb from mouse (Sigma-Aldrich, 1:3000 dilution), anti-α-tubulin mAb from mouse (GeneTex, 1:5000) and anti-Vif mAb from mouse (NIH AIDS Reagent Program #319, 1:2000 dilution) as primary antibodies. Cy3-labeled goat-anti-mouse mAb (GE Healthcare, 1:3000 dilution) was used as a secondary antibody to detect the signal. The fluorescent signal was detected and visualized with Typhoon RGB Biomolecular Imager (GE Healthcare).

One hundred nanograms of FLAG-A3G-pcDNA or HA-A3G-pcDNA variants were cotransfected with 200 ng of either Vif-pcDNA or pcDNA3.1(+) empty vector into human embryonic kidney 293T cells (American Type Culture Collection) in 12-well plates (CELLTREAT) using the X-tremeGENE 9 DNA Transfection Reagent (Roche). Cells were incubated at 37°C in 5.0% CO₂. At 24 hours after transfection, 2 μM N-carbobenzyloxy-l-leucyl-l-leucyl-l-leucinal (Sigma-Aldrich) or dimethyl sulfoxide control was added. At 48 hours after transfection, the cells were washed once with phosphate-buffered saline and lysed in radioimmunoprecipitation assay buffer with 1× cOmplete Protease Inhibitor (Roche). The lysates were then subjected to Western blot with anti-FLAG M2 monoclonal antibody (mAb) from mouse (Sigma-Aldrich), anti–α-tubulin mAb from mouse (GeneTex), and anti-Vif mAb from mouse (National Institutes of Health AIDS Reagent Program #319) as primary antibodies. Cy3-labeled goat anti-mouse mAb (GE Healthcare) was used as a secondary antibody to detect the signal. The fluorescent signal was detected and visualized with Typhoon RGB Biomolecular Imager (GE Healthcare).