Anti hiv 1 vif 319

Western blotting analysis was performed as described previously [37 (link),38 (link)]. Briefly, pro-viral clones or pcNLmini-RI-based clones were transfected into HEK 293T cells by Lipofectamine2000, and unless otherwise noted, on day 1, post-transfection cell lysates were prepared with 1 × TNE buffer. Analyses of Vif and A3G expression levels in virions and cells were carried out similarly as described previously [43 (link),44 (link)]. Briefly, HEK 293T cells were co-transfected with pro-viral clones along with a FLAG-tagged A3G expression vector by the calcium phosphate coprecipitation method. On day 1 post-transfection, virions were collected by ultracentrifugation, and the lysates of virions and cells were prepared with 1 × TNE buffer. Anti-HIV-1 Vif 319 (catalog no. ab66643; Abcam, Tokyo, Japan), anti-Gag-p24 (#3537, NIH Research and References Reagent Program), anti-FLAG (FLA-1, MEDICAL & BIOLOGICAL LABORATORIES), and anti-β-actin clone AC-15 (Sigma-Aldrich, Burlington, MA, USA) antibodies were used for immunoblotting analyses.

Proviral clones were transfected into 293T cells, and on day 1 post-transfection, cell lysates were prepared with 1 × TNE buffer (Nomaguchi et al., 2014 (link), 2016 (link)). Western blot analysis was carried out as previously described using anti-Vif [anti-1Q (Akari et al., 1999 (link)), anti-HIV-1 HXB2 Vif (catalog no. 2221; NIH Research and References Reagent Program) or anti-HIV-1 Vif 319 (catalog no. ab66643; Abcam)] and anti-β-actin clone AC-15 (Sigma–Aldrich) antibodies (Nomaguchi et al., 2014 (link), 2016 (link)).