Fig 7.

TRIM27-USP7 regulation of RIP1 ubiquitination in the presence of TNF-α and CHX. (A) Knockdown of USP7 using siRNA in immortalized MEF. Three different sequences of mouse USP7 siRNA were transfected, and cell lysates were analyzed by Western blotting with anti-USP7 (top) or anti-tubulin (bottom) antibodies. (B) USP7 is involved in TNF-α–CHX-induced apoptosis. Cell viability was examined as described for Fig. 1H using control immortalized MEFs and USP7 knockdown cells. Experiments were repeated three times, and average values relative to the nontreated cells with SD are shown. **, P < 0.01. (C) Cleaved caspase-3 was determined by immunoblot analysis of MEFs lysates prepared at 2.5 h after TNF-α–CHX addition. (D and E) Ubiquitination of endogenous RIP1 after TNF-α–CHX treatment. Immortalized WT, Trim27^−/− (D) or USP7 knockdown (E) MEFs were treated with TNF-α (10 ng/ml) and CHX (1 μg/ml) for the indicated times. Endogenous RIP1 was immunoprecipitated and analyzed by immunoblotting with anti-Ub (top) or anti-K63-linked Ub (bottom) antibody. Lysates were also analyzed by Western blotting with anti-RIP1 or anti-USP7 antibodies.