Rabbit anti vps28 antibody

293T cells (2.2 × 10⁶) were seeded into T25 flasks and transfected with VPS28-specific RNAi or control RNAi (Invitrogen), 1–2 μg of LYPxNL-abrogated (YP-) mutant HIV-1 proviral DNA together with expression vectors for RNAi Resistant (RR) wild-type (WT) or mutant VPS28, (TM) or an empty vector control. Twenty-four hours after transfection, culture supernatants were harvested as previously described in^{68 (link)} and virions were then pelleted through 20% sucrose cushions. Cells were collected and lysed in 0.5% lysis buffer (RIPA Buffer) (140 mM NaCl, 8 mM Na2HPO4, 2 mM NaH2PO4, 1% Nonidet P-40 [NP-40], 0.5% sodium deoxycholate, 0.05% sodium dodecyl sulfate [SDS]) and Complete Protease Inhibitor Cocktail (Roche). Isolated virions and viral proteins in cell lysates were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) and Western blotting for viral proteins with a goat anti-HIV NC (a generous gift from Robert Gorelick) and a mouse anti-CA antibody (AIDS Repository cat# clone 183-H12–5C). Host proteins were detected by Western blotting with mouse anti-tubulin and VPS28 expression was verified with a rabbit anti-VPS28 antibody (Sigma).