Ubiquitination of ESCRT-I is not sufficient for HIV-1 release. (A and B) 293T cells were co-transfected with HA-Ub plasmid and Flag-tagged version of ESCRT-I components [TSG101 (2.5 μg), VPS28 (800 ng), VPS37B (1.7 μg), MVB12B (800 ng); these amounts express comparable levels of ESCRT-I proteins] alone, or in addition to the following plasmids: either Gag-Strep (1.5 μg) (lanes 2, 7, 12) or Gag-DUbStrep alone (1.5 μg) (lanes 3, 8, 13) or in combination with the Nedd4-2s expression plasmid (150 ng) (lanes 4, 9, 14) and with Gag-DUb*Strep alone (1.5 μg) (lanes 5, 10, 15). Sequential centrifugations were performed to separate membrane-enriched P100 fractions from which Gag molecules were immunoprecipitated using Strep-Tactin beads. The protein content of captured complexes (A), input (lower left and center panels) and virus (lower right panel) fractions were analyzed by WB with the indicated antibodies. (B) shows a darker exposure of samples analyzed in lanes 6–10).