Role of APH-2 in HIV-1 release. (A) Effect of APH-2 on pGag-EGFP: 293T cells were transfected with HXB2 pGag-EGF (1 µg) only and co-transfected with HXB2 pGag-EGF (1 µg), along with increasing amounts of plasmid-encoding HA-APH-2 (1–3 µg). Western blots were performed to examine cell-free virus-like particle production (Panel 1), cellular Gag (pr55-EGFP) expression (Panel 2) and APH-2 expression (Panel 3). Bottom panel: Cellular beta actin expression represents the loading control. (Amount of plasmid used is represented in figure as − = 0 μg; + = μg; ++ = 2 μg; +++ = 3 μg; ++++ = 4 µg). The graph below shows densitometric analysis of Western blot performed using Image J software, and relative cellular protein expression normalized to β-actin. (B) HIV-1 virus release efficiency: Densitometric analysis was performed using NIH Image J software and a graph was plotted for average percent change in Gag expression. The efficiency of virus release was calculated as the amount of virion-associated mature capsid protein, p24, as a fraction of the total amount of Gag (virion-associated p24 plus all the cellular precursors of Gag) relative to pGag-EGFP, which was arbitrarily set at 100.