Cell and supernatant samples were separated by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) on 11% gel for p24Gag and 15% gels, according to the size of the target proteins, at 27 mA per gel. Proteins were transferred onto nitrocellulose membrane overnight at 16 V and blocked for 1 h with 1% fat-free milk in 0.1% Tween/PBS. Membranes were incubated with primary antibodies at room temperature for at least 1 h.
The following primary antibodies were used: anti-HIV-1 Gag antibody (mouse hybridoma 183-H12-5C supernatant—kind gift from Prof. M. Malim), anti-GFPT1 (Abcam AB125069), anti-KGA (Proteintech 20170-1-AP), anti-GAC (Proteintech 19958-1-AP), anti-PPAT (OriGene TA504769S) and anti-HSP90 (Santa Cruz sc-7947). After incubation with primary antibodies, membranes were washed and incubated with secondary antibodies 680RD goat anti-mouse 926-68070 and 800CW goat anti-rabbit 926-32211 (both LI-COR) at room temperature for 1 h. Membranes were then washed again before visualization with a quantitative LI-COR imager.
The following primary antibodies were used: anti-HIV-1 Gag antibody (mouse hybridoma 183-H12-5C supernatant—kind gift from Prof. M. Malim), anti-GFPT1 (Abcam AB125069), anti-KGA (Proteintech 20170-1-AP), anti-GAC (Proteintech 19958-1-AP), anti-PPAT (OriGene TA504769S) and anti-HSP90 (Santa Cruz sc-7947). After incubation with primary antibodies, membranes were washed and incubated with secondary antibodies 680RD goat anti-mouse 926-68070 and 800CW goat anti-rabbit 926-32211 (both LI-COR) at room temperature for 1 h. Membranes were then washed again before visualization with a quantitative LI-COR imager.