ESCRT requirements for HIV-1 and EIAV release and infectivity. Viral titers (top panel, note log scale), and western blots showing levels of virion-associated Gag proteins (panel 2, “Virus”) and the designated intracellular viral and cellular proteins in 293T cells (lower panels, “Cell”) expressing HIV-1 (A) or EIAV (B), with positions of molecular weight markers shown at right. Virus-producing cells were treated with control siRNAs (lanes 1 and 2) or siRNAs that depleted ALIX (lane 3), TSG101 (lane 4), CHMP2A and CHMP2B (lane 5), CHMP4A and CHMP4B (lane 6), or VPS4A and VPS4B (lane 7). Bottom panels show GAPDH loading controls and cellular levels of the designated ESCRT protein following treatment with the control siRNA (equivalent to control lanes 1 and 2) or a specific siRNA (equivalent to the lane designated beneath the blot). Band intensities were integrated for the shown blots to quantify levels of virus release (panel 2, CA band intensities) and target protein depletion (bottom panels), and values are relative to the average intensities of the two control lanes. Error bars in the top panel show the standard deviation from the mean of 3–6 independent repetitions of the experiment.