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. 2012 Sep 4;287(43):36617–36622. doi: 10.1074/jbc.M112.407130

FIGURE 4.

FIGURE 4.

Deubiquitination of NLRP3 is required for its priming and activation. A, B, and F, anti-ubiquitin (UB) antibody (upper panels) and anti-NLRP3 antibody (lower panels) immunoblots of anti-FLAG immunoprecipitates from Nlrp3-KO and N1-8 macrophages (A, first and second lanes, respectively) and N1-8 macrophages stimulated with LPS, ATP, or LPS plus ATP in the presence or absence of the deubiquitination inhibitors PR-619 and WP1130 (B) and the antioxidant NAC (F) as indicated. C, immunoblots of caspase-1 in the culture supernatants of N1-8 and NG5 macrophages stimulated with LPS plus ATP (N1-8) or ATP (NG5) in the presence or absence of PR-619 or WP1130 as indicated (upper panels). The lower panels show immunoblots of caspase-1 in the cell lysates of the same macrophages. D, anti-ubiquitin antibody (upper panel) and anti-NLRP3 antibody (middle panel) immunoblots of anti-FLAG immunoprecipitates from 293T-caspase-1-ASC (293T-CA) and 293T-caspase-1-ASC-NLRP3 (293T-CAN) cells stimulated with nigericin (Nig) for 60 min or left untreated (Un). The lower panel shows a caspase-1 immunoblot in the culture supernatants of the same cells. E, immunoblot of caspase-1 in cell lysates of 293T-caspase-1-ASC-NLRP3 cells treated with nigericin in the presence or absence of PR-619 as indicated. Procasp-1, procaspase-1.