Figure 3

Intracellular localization of IFNβ-treated HERC5 and HIV-1 Gag. A, Localization of endogenous HERC5 (red) in IFNβ-treated (500 units/ml for 16 hours) PBMCs (scale bar = 5 μm), Jurkat cells (scale bar = 5 μm), and murine dendritic cells (scale bar = 10 μm). Images shown are representative of at least 2 independent experiments. B, Localization of endogenous ribosomes (green) and HERC5 (red) in control Jurkat cells or IFNβ-treated Jurkat cells (scale bar = 5 μm). Localization of Gag alone (red) or HERC5 (green) +/- CS (C), or co-expression of Gag and HERC5 + CS (D) in U2OS cells 48 hours after transfection. Scale bars = 20 μm. Images shown are representative of at least 3 independent experiments. E, HeLa cells were co-transfected with pR9, pUbe1L, pUbcH8, and pMyc-ISG15 with or without pFlag-HERC5 or pFlag-HERC5-C994A. Gag was immunoprecipitated under non-denaturing conditions using anti-p24CA. Precipitated proteins were resolved using SDS-PAGE and subjected to Western blotting using anti-Flag or anti-p24CA. 10% of the input lysates was subjected to Western blotting using anti-p24CA and anti-β-actin on the same blot. HERC5 protein was undetectable in the 10% input lysates.