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. 2019 May 31;9(14):4208–4220. doi: 10.7150/thno.33803

Figure 4.

Figure 4

USP5 stabilizes TUFM through deubiquitination. A. Myc-USP5-WT, Myc-USP5-C335A and Flag-TUFM were co-transfected into HEK293T cells. Twenty-four hours later, cells were prepared for immunoblotting analysis using anti- Flag, Myc and GAPDH antibodies. B. HCT116 cells were transfected with increased amounts of plasmids Myc-USP5-WT and Myc-USP5-C335A, and then analyzed by immunoblotting with antibodies against TUFM, Myc and GAPDH. C. After infected with lentiviruses expressing shUSP5#1, shUSP5#2, shUSP5#3 or control for 3 days, HCT116 cells were lysed and analyzed by immunoblotting against USP5 and TUFM. GAPDH was used as a loading control. D. After transfected with Myc-USP5 or control vector for 24 hours, HCT116 cells were exposed to CHX for indicated time and analyzed for TUFM and Myc-USP5 levels by immunoblotting. GAPDH was used as a loading control. E. Quantitative and statistical analyses of data from Figure D (mean +/- SD). F. Following infected with lentiviruses expressing shUSP5#3 or shNC for 36 hours, HCT116 cells were treated with 10 μM of MG132 for 12 hours and then analyzed for the levels of TUFM and USP5 by immunoblotting. GAPDH was used as a loading control. G. Quantitative and statistical analyses of data from Figure F (mean +/- SD). H. Plasmids expressing Myc-USP5-WT, Myc-USP5-C335A, Flag-TUFM or HA-Ub-K48 were transfected into HEK293T cells for 24 hours. The cells were then lysed, immunoprecipitated with anti-Flag antibody, and immnublotted with anti-HA antibody as indicated. The cell lysates were also directly immunoblotted with anti- Flag and Myc antibodies. GAPDH was used as a loading control. I. HCT116 cells were transfected with plasmids Flag-TUFM, HA-Ub-K48, or infected with lentiviruses expressing shUSP5#1, shUSP5#3 or shNC for 48 hours. After treated with 20 μM of MG132 for 6 hours, the cells were lysed, immunoprecipitated with anti-Flag antibody, and immunoblotted with anti-HA antibody to detect the ubiquitination of TUFM. The cell lysates were also directly immunoblotted with antibodies against Flag, USP5 and GAPDH.