Fig. 4.

IL-1β- or Tax-induced ubiquitinated TRAF6 is deubiquitinated by USP20. (A) HEK293T cells were transfected as indicated with Flag-TRAF6, HA-Ub, and USP20. Cells without IL-1β treatment (lanes 1 and 2) and cells treated with IL-1β for 8 h (lanes 3 and 4) were subjected to immunoprecipitation (IP) followed by immunoblotting (IB) to detect ubiquitinated TRAF6 (anti-HA; top panel). WCE, whole-cell extract. (B) HEK293T cells were transfected as indicated with Flag-TRAF6, HA-Ub, and siRNA specific for USP20. Cells without treatment (0 h; lanes 1 and 2) and cells treated with IL-1β for 8 h (lanes 3 and 4) were immunoprecipitated and detected by immunoblotting for ubiquitinated TRAF6 (anti-HA; top panel). (C) HEK293T cells were transfected as indicated with Tax, Flag-TRAF6, HA-Ub, and USP20 (lanes 2 to 5). Cell lysates were immunoprecipitated and immunoblotted for detection of ubiquitinated TRAF6 (anti-HA; top panel). Total amounts of transfected DNA or transfected siRNA were equalized by the addition of empty vector or control siRNA, respectively. Immunoprecipitations were performed using anti-Flag antibody. Ubiquitinated proteins were detected by immunoblotting with anti-HA antibody. Anti-Flag (for TRAF6), anti-USP20, anti-Tax, anti-tubulin, and anti-actin antibodies were used to verify the respective proteins in immunoblottings. To normalize the amount of ubiquitinated TRAF6, signals of ubiquitinated TRAF6 and total TRAF6 in the immunoblots were quantified by densitometry, and relative levels of normalized ubiquitinated TRAF6 were calculated. The results are graphed in the middle of each panel.