Additional file 6: of A single-residue change in the HIV-1 V3 loop associated with maraviroc resistance impairs CCR5 binding affinity while increasing replicative capacity

Figure S4. Western blot analysis of gp120 and Gag p24 expression on MVC-Sens, MVC-Res, MVC-Sens(V3R) and MVC-Res(V3S) viral particles. Three hundred ng Gag p24 of the different viral isolates were solubilized in lysis buffer (100 mM (NH4)2SO4, 20 mM Tris-HCl (pH 7.5), 10 % glycerol, and 1% Triton X-100) and then loaded onto Biorad CriterionTM XT 4-12% Bis-Tris Gels under reducing conditions. Virus-associated gp120 and p24 were visualized using a sheep anti-HIV-1 gp120 polyclonal antibody (AALTO Bio Reagents LTD.) and a mouse anti-HIV-1 p24 monoclonal antibody (clone 183-H12-5C) (NIH AIDS Reagent Program), respectively, and then with the appropriate HRP-conjugated secondary antibodies (VECTOR). Bands were developed by enhanced chemiluminescence (Thermo Scientific) and were quantified using a LAS-1000 CCD camera (Image Gauge Software, Fuji Film Co., Tokyo, Japan). The figure shows that the viral isolates have gp120/p24 ratios R that are in the same range (R = 1, 1.15, 1.24 and 0.81 for MVC-Sens, MVC-Res, MVC-Sens(V3R) and MVC-Res(V3S), respectively). A representative experiment out of two is shown