Radioimmunoprecipitation Assay for HIV-1 Proteins

Radioimmunoprecipitation assays were performed as described previously.^{[22 (link)]} Briefly, 293T cells were transfected with pNL4–3 by means of Lipofectamine 2000. At 24 h post-transfection, cells were starved in Met/Cys-free medium for 30 min and then metabolically labeled with [³⁵S]Met/Cys-Pro mix (PerkinElmer, Waltham, MA, USA) for 2 h. Cells were treated with compound 1 throughout the transfection and labeling period. Viruses were collected by ultracentrifugation at 75 000 g for 45–60 min. Cell and virus pellets were resuspended in Triton X-100 lysis buffer (300 mm NaCl, 50 mm Tris-HCl [pH 7.5], 0.5% Triton X-100, 10 mm iodoacetamide, and protease inhibitor cocktail tablets [Roche, Indianapolis, IN, USA]), followed by preclearance for 1–2 h and immunoprecipitation with HIV-Ig (NIH AIDS Research and Reference Reagent Program, cat. #3957). Immunoprecipitated proteins were separated on gels containing 12% polyacrylamide by SDS-PAGE, dried, exposed to a phosphorimager plate (Fujifilm, Stamford, CT, USA), and quantified with Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA).