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. 2019 Aug 9;8:e48318. doi: 10.7554/eLife.48318

Figure 4. USP49 directly interacts with A3G and deubiquitinates the K48-linked ubiquitination of A3G.

Figure 4.

(A) HEK293T cells were co-transfected with plasmids expressing USP49-GFP or A3G-RFP. After 48 hr, the cells were fixed with 4% paraformaldehyde, and localization of USP49 and A3G was detected using confocal microscopy. (B) HEK293T cells were co-transfected with plasmids expressing Flag-USP49 or HA-A3G, or the indicated vectors. The cell lysates were immunoprecipitated using Flag beads and analyzed by immunoblotting with the indicated antibodies. (C) Hela cells were co-transfected with plasmids expressing HA-GFP or HA-A3G. The cell lysates were immunoprecipitated using HA beads and analyzed by immunoblotting with the indicated antibodies. (D) HEK293T cells were co-transfected with plasmids expressing HA-USP49 and Flag-A3G. The cell lysates were immunoprecipitated using HA beads and then treated with RNase A (20 ug/mL) for 1 hr. After that, samples were analyzed by immunoblotting with the indicated antibodies. (E) HEK293T cells were co-transfected with plasmids expressing USP49-HA, Vif-HA, Ub-HA, or A3G-Flag. Cells were treated with 10 uM MG132 for 24 hr before harvest. After 48 hr post-transfection, the cells were subjected to the denaturing immunoprecipitation using an anti-Flag beads followed by immunoblot analysis using the indicated antibodies. (F) HEK293T cells were transfected with the indicated plasmids expressing HA-ubiquitin, Flag-A3G, HA-Vif, or HA-USP49. After 24 hr, the cells were treated with 10 μM MG132 for 24 hr; the indicated types of ubiquitination were detected and quantified by the denaturing immunoprecipitation and western blotting.