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. 2019 Mar 18;294(18):7283–7295. doi: 10.1074/jbc.RA118.007257

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© 2019 Ali et al.

Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc.

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Figure 4. — A, TZM-bl cells were co-transfected with Tat along with CHIP- or CHIPΔU-expressing constructs, and 36 h post-transfection, cells were lysed, and luciferase activity was measured. As a transfection control, GFP was used, and the luciferase readings were normalized with GFP fluorescence and plotted. B, pNL4-3Luc.R⁻E⁻ was transfected with CHIP- or CHIPΔU-expressing plasmids into TZM-bl cells, and luciferase activity was measured and plotted after normalization with GFP. C, pNL4-3 was co-transfected with increasing doses of CHIP in HEK-293T cells, and after 36 h, the Gag was immunoblotted. The blot is representative of three different independent experiments. D, increasing doses of CHIPΔU were co-transfected with 10 μg of pNL4-3. The Gag level was monitored by immunoblotting after 36 h of transfection. The blot is representative of three different independent experiments. E, Jurkat cells were transfected with pNL4-3 and CHIP or CHIPΔU, and the Gag protein levels were measured by immunoblotting after 48 h of transfection. F, HEK-293T cells were transfected with pNL4-3 and CHIP or A3G, and the Gag protein levels were measured by immunoblotting. The blot is representative of three different independent experiments. G, TZM-bl cells were transfected with CHIP, and after 24 h, the cells were infected with VSVG-pseudotyped HIV-1. Cells were further cultured for 24 h followed by staining for β-gal activity as described under “Experimental procedures.” The image is representative of three different independent experiments. H, β-gal staining intensity was measured by ImageJ and plotted. I, HEK-293T cells were transfected with CHIP, and after 24 h, the cells were infected with VSVG-pseudotyped HIV-1. Cells were cultured for 24 h, and immunoblotted for Gag. The blot is representative of three different independent experiments. J, Gag level was measured with ImageJ and plotted. K, HEK-293T cells were transfected with CHIPΔU, and after 24 h, the cells were infected with VSVG-pseudotyped HIV-1. Cells were further cultured for 24 h and immunoblotted for Gag. The blot is representative of three different independent experiments. L, Gag level was measured with ImageJ and plotted. The blot is representative of three different independent experiments.