Fig. 6.

USP7 stabilizes c-Myc levels by antagonizing TRIM32 ubiquitination activity. a Cycloheximide treatment of HEK293T cells transfected for 48 h with plasmids expressing FLAG-USP7 or an empty vector as control. Endogenous c-Myc levels were analyzed at 0, 30, 90 and 150 min after cycloheximide treatment by western blot with an anti-c-Myc antibody. GAPDH western blots were used as loading control. b Quantification of endogenous c-Myc levels in the presence or absence of overexpressed USP7 as shown in (a), normalized to their respective time point 0 (mean ± SEM; N = 5 independent experiments; Mann–Whitney U test or t test, **p < 0.01). c c-Myc in vitro deubiquitination assay using recombinant proteins. E1 and E2 (UbcH5a) enzymes were incubated with the indicated components followed by 2 × IP of c-Myc with an anti-c-Myc antibody. Ubiquitination of c-Myc was detected by immunoblotting with an anti-FLAG antibody, directed against the FLAG-tag of ubiquitin, as well as with an antibody directed against mono-and-polyubiquitinylated conjugates (anti-(Ub)_n). d Quantification of polyubiquitinated c-Myc levels in FLAG and (Ub)_n blots shown in (c), normalized to the control (mean ± SEM; N ≥ 3 independent experiments; Mann–Whitney U test or t test, **p < 0.01; *p < 0.05). WB western blot, CHX cycloheximide, IP immunoprecipitation