A Mapping of interaction regions between MARCH7 and NLRP3. B IP analysis of interaction regions between MARCH7 and NLRP3. C Western blot analysis of NLRP3 expression after overexpression of MARCH7 and another 6 h of incubation with MG132 and E64. D Western blot analysis of NLRP3 expression and detection of NLRP3 ubiquitination by IP. AML12 and HepG2 cell lines were transfected with HA-NLRP3 alone or together with increasing amounts of Flag-MARCH7. Twenty-four hours later, these cells were treated with or without 20 μM MG132 for another 12 h. Then, the cells were collected for IP with an anti-HA antibody to detect NLRP3 ubiquitination. E The cell lines were transfected with HA-NLRP3 alone or together with the indicated Flag-MARCH7 constructs; WT wild type, mut mutant (W589A/I556A). Thirty-six hours later, these cells were prepared for detection of NLRP3 ubiquitination using an IP assay. The data are presented as the mean ± S.D. of three independent experiments. A p value of < 0.05 was considered statistically significant; and *p < 0.05, **p < 0.01, ***p < 0.001 assessed via a ANOVA with the Bonferroni post hoc test for comparisons among more than two groups (C); ns not significant.