Fig. 4. gp78 mediates mixed ubiquitination of NLRP3.
A, B BMDMs from Lyz2-cre-gp78f/f mice were lentivirally transduced with Phage empty vector, Phage-FLAG gp78 or Phage-FLAG-gp78 C356G, then primed with 500 ng/ml LPS for 4 h followed by stimulating with ATP (2 mM) or nigericin (10 μM) for 30 min. Supernatants (SN) were analyzed by ELISA for IL-1β release (A, n = 4). SNs and cell extracts (Lysate) were also analyzed by immunoblotting (B). C Peritoneal macrophages from gp78f/f and Lyz2-cre-gp78f/f mice were primed with or without LPS (500 ng/ml) for 4 h. Cell lysates were immunoprecipitated with anti-NLRP3 antibody or anti-mouse IgG and then immunoblotted with the indicated antibodies. D Peritoneal macrophages from wild-type and CD68-gp78 mice were primed with LPS (500 ng/ml) for 4 h. NLRP3 of peritoneal macrophages was immunoprecipitated for ubiquitination analysis. E gp78f/f and Lyz2-cre-gp78f/f mice were intraperitoneally injected with or without LPS (20 mg/kg body weight) for 4 h. NLRP3 protein of peritoneal cells was immunoprecipitated for ubiquitination analysis. F HEK293T cells were transfected with Flag-tagged NLRP3, HA-tagged Ub, along with different doses of Myc-tagged gp78. Cell lysates were immunoprecipitated with anti-Flag antibody and then immunoblotted with the indicated antibodies. G HEK293T cells were transfected with Flag-tagged NLRP3 and HA-tagged Ub, along with Myc-tagged gp78 and its catalytically inactive mutants (C356G) or Myc-tagged Hrd1. Cell lysates were immunoprecipitated with anti-Flag antibody and then immunoblotted with the indicated antibodies. H The in vitro ubiquitination reaction mixture contains E1, E2, Ub, GST-NLRP3 ΔLRR, and gp78 or gp78 C356G as indicated. After incubation at 30 °C for 30 min, the mixture was assessed by immunoblotting with the indicated antibodies. I HEK293T cells expressing Flag-tagged NLRP3, Myc-tagged gp78, and HA-tagged Ub, HA-tagged K48-Ub, or HA-tagged K63-Ub were immunoprecipitated with anti-Flag antibody for ubiquitination. J HEK293T cells expressing Flag-tagged NLRP3, HA-tagged Ub, and Myc-tagged gp78 or V5-tagged BRCC3 as indicated were immunoprecipitated with anti-Flag antibody for ubiquitination. K HEK293T cells expressing Flag-tagged NLRP3, Myc-tagged gp78, HA-tagged Ub, or HA-tagged K48/K63R Ub as indicated were immunoprecipitated with anti-Flag antibody for ubiquitination. *p < 0.05, **p < 0.01, ***p < 0.001. Values are mean ± SD (A). Data are representative of three independent experiments.