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. 2015 Jul 23;290(36):22184–22192. doi: 10.1074/jbc.M115.675207

FIGURE 4.

FIGURE 4.

ERManI inhibits HIV-1 Env expression. A and B, 293T cells were transfected with pNL4-3, and a mammalian vector expressing indicated human or murine (m-) proteins, respectively. After 48 h, protein expression was determined by Western blotting using the indicated antibodies. C, titration of the ERManI anti-Env activity. 293T cells were transfected with pNL4-3 and an ERManI expression vector at indicated ratio, and protein expression was determined by Western blotting. D, 293T cells were transfected with 2.0 μg of pNL4-3 and 1.0 μg of ERManI expression vectors. Cells were treated with 25 μm lactacystin or 5 μm KIF for 4 h, or untreated (control, (Ctrl)). Protein expression was determined by Western blotting using indicated antibodies. E, ERManI reduces HIV-1 infectivity. HIV-1 luciferase reporter viruses were produced after transfection of 293T cells with the HIV-1 proviral vector pNL-Luc and a WT ERManI, its catalytically inactive mutant E330A, or a control (Ctrl) vector. Viruses were normalized by p24Gag ELISA and used to infect GHOST cells. After 48 h, cells were lysed, and viral infectivity was determined by measuring the Luc activity. Error bars represent S.E. from three independent experiments.