αmouse irdye 680lt

For immunoblotting assays, the indicated cell line was plated at a seeding density of approximately 250,000 cells per well in a 12 well plate. The next day, cells were either transfected with 250 ng of the indicated Vif construct, or, transduced with the indicated virus and allowed to incubate for 48 h. After 48 hours post-transfection/transduction, cells were collected into 1.5 mL tubes and centrifuged at 500 × g for 10 minutes. Cell pellets were resuspended in 100 mL RIPA buffer (10 μM Tricl-Cl [pH 8.0], 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, and 140 mM NaCl) and 10% of the total cell lysate samples were combined with 2.5x SDS-PAGE loading buffer. Samples were separated by a 4%–20% gradient SDS-PAGE gel and transferred to PVDF-FL membranes (Millipore). Membranes were blocked in blocking solution (5% milk + PBS supplemented with 0.1% Tween20) and then incubated with primary antibody diluted in blocking solution. Secondary antibodies were diluted in blocking solution + 0.01% SDS. Membranes were imaged with a LICOR Odyssey instrument or film. Primary antibodies used in these experiments were αActin (Cell Signaling #4970) and αVif (NIH Reagent Program #6459). Secondary antibodies used were αrabbit IRdye 800CW (LICOR 827–08365), αmouse IRdye 680LT (LICOR 925–68020), αrabbit HRP (Cell Signaling 7074P2), and αmouse HRP (Cell Signaling 7076P2).