Figure 2: TRIM31 promotes proteasomal degradation of NLRP3.

(a) Immunoblot analysis of extracts (upper panel) or RT-PCR analysis (lower panel) of mouse peritoneal macrophages silenced of TRIM31. (b) Immunoblot analysis of extracts (upper panel) or RT-PCR analysis (lower panel) of mouse peritoneal macrophages silenced of TRIM31, then stimulated for various times with LPS. (c) Immunoblot analysis of extracts from HEK293T cells transfected with increasing amount of TRIM31 expression plasmid. (d) Immunoblot analysis of extracts from TRIM31^+/+ or TRIM31^−/− mouse peritoneal macrophages, then stimulated for various times with LPS. (e) Immunoblot analysis of NLRP3 expression from TRIM31^+/+ or TRIM31^−/− mouse peritoneal macrophages, then stimulated with LPS, IL-1β, poly(I:C), TNF-α or PGN for 8 h. (f,g) Immunoblot analysis of extracts from TRIM31^+/+ or TRIM31^−/− mouse peritoneal macrophages stimulated with LPS for 4 h, and then treated for various times with cycloheximide (CHX). NLRP3, ASC, Caspase-1 and NLRC4 expression levels were quantitated by measuring band intensities using ‘ImageJ’ software. The values were normalized to actin (g). **P<0.01. (mean and s.d. of three samples in g, Student's t-test). (h) Immunoblot analysis of extracts from HEK293T cells transfected with Myc-NLRP3 and Flag-TRIM31 expression plasmid then treated with MG132 (10 μM) or chloroquine (10 μM) for 4 h. (i) Immunoblot analysis of extracts from HEK293T cells transfected with Myc-NLRP3 and Flag-TRIM31 expression plasmid then treated with 3-MA as indicated for 4 h. (j) Immunoblot analysis of NLRP3 expression from TRIM31^+/+ or TRIM31^−/− mouse peritoneal macrophages stimulated with LPS for 4 h, together with DMSO or MG132 (10 μM) treatment for 4 h. Similar results were obtained in three independent experiments.