Analysis of Env incorporation into viruses pseudotyped with CXCL12-sensitive and CXCL12-resistant Envs.

A Western blot analysis of gp120 and p24 expression into viruses pseudotyped with early (SENS) Envs (#1, 16, 29, 36 and 44), late (RES) Envs (#6, 28, 32, 41 and 47) or NL4-3 Env. Three hundred ng of Gag p24 of the different viruses were solubilized in lysis buffer containing XT sample buffer (Biorad), Invitrogen NuPAGE sample reducing agent and 1% Triton X-100, incubated for 5 min at 70°C and then loaded onto Biorad Criterion XT 4–12% Bis-Tris gels under reducing conditions and then transferred to nitrocellulose membrane. Membranes were blocked with Odyssey blocking buffer (Li-COR) (for p24 detection) or TBS containing 5% BSA and 0.05% NaN3 (for gp120 detection) and then incubated overnight at 4°C with a sheep anti-HIV-1 gp120 polyclonal antibody (clone D7324, Aalto Bio Reagents) or for 1 h at RT with a mouse anti-HIV-1 p24 mAb (clone 749140, R&D Systems). Membranes were then incubated with the following species-specific secondary antibodies: DyLight 800-conjugated donkey Anti-Sheep IgG (Novusbio) and IRDye 800CW-conjugated goat Anti-Mouse (Li-COR) (dilution: 1/10,000). Signals were detected with a Li-COR Odyssey scanner and quantified using ImageStudioLite software. Arrow indicates gp120 bands. As control, thirty ng of purified gp120 #28 were also immunodetected. A representative experiment out of two independent experiments with distinct virus preparations is shown. B Band intensity ratios of gp120 to p24, normalized to NL4-3. Means ± SEM of two independent experiments are shown. The color code is the same as in Fig 1A. Statistics: Mann-Whitney test.

(PPTX)