Figure 6. AMOT p130 stimulates release of HIV-1 from HeLa and Jurkat T cells.

(A) Left panels are western blots showing HeLa cellular levels of endogenous AMOT p130 and exogenous HA-AMOT p130 (panel 1, anti-AMOT), endogenous GAPDH (panel 2, anti-GAPDH, loading control) and HIV-1 Gag proteins (panel 3, anti-MA and anti-CA). HeLa cells were co-transfected with expression vectors for HIV-1 (lanes 1–5), and with increasing concentrations of an expression construct for HA-AMOT p130 (0–4 μg DNA in lanes 1–5). Right panels show corresponding levels of extracellular virion-associated Gag proteins (upper panel, western blot, anti-MA and anti-CA) and viral titers (lower panel), n = 4. (B) Levels of extracellular virion-associated Gag proteins (upper panel, western blot, anti-MA and anti-CA) and viral titers (lower panel). Jurkat T cells were co-transfected with expression vectors for HIV-1 (lanes 1–5), together with a control vector (lane 1) or expression vectors for wild type HA-AMOT p130 (lanes 2 and 3, 5 and 10 µg DNA respectively) or an HA-AMOT p130_ΔPPXY protein with inactivating point mutations in the three PPXY motifs (lanes 4 and 5, 5 and 10 µg DNA respectively), n = 3.

DOI: http://dx.doi.org/10.7554/eLife.03778.015

Figure 6—figure supplement 1. HeLa cells express little or no AMOT.

Western blots showing endogenous AMOT p130 and p80 expression levels in 293T cells (lanes 1–4) and in HeLa cells (lanes 5–8). Increasing numbers of cell equivalents were added as indicated (ranging from 1.5 × 10⁵ cells in lanes 1 and 5 to 1.2 × 10⁶ cells in lanes 4 and 8). Upper and lower panels show lighter and darker exposures of the same blot. Image is representative of two independent repetitions of this experiment.

DOI: http://dx.doi.org/10.7554/eLife.03778.016