Figure 3: TRIM31 interacts with NLRP3.

(a) NLRP3 and Flag-tagged TRIM31 were obtained by in vitro transcription and translation. Interaction between TRIM31 and NLRP3 was assayed by mixing TRIM31 and NLRP3 together followed by IP with Flag antibody and immunoblot analysis with NLRP3 antibody. (b) Co-immunoprecipitation of endogenous TRIM31 with endogenous NLRP3 from mouse peritoneal macrophages stimulated with LPS for indicated time periods. (c) HEK293T cells expressing Flag-TRIM31 and Myc-NLRP3, Myc-Caspase-1 or Myc-ASC were lysed. Co-immunoprecipitation of Flag-TRIM31 with Myc-NLRP3 from HEK293T cells. *Non-specific band. (d) HEK293T cells transfected with GFP-TRIM31 and Myc-NLRP3 were fixed and incubated with a secondary antibody conjugated to Alexa Fluor 568. Colocalization between TRIM31 and NLRP3 was examined by Confocal microscopy. (e) Schematic diagram of TRIM31 and its truncation mutants. (f) Flag-tagged TRIM31 or its mutants and Myc-NLRP3 were individually transfected into HEK293T cells. The cell lysates were immunoprecipitated with an anti-Flag antibody and then immunoblotted with the indicated antibodies. (g) Schematic diagram of TRIM31 and its truncation mutants. (h) Myc-tagged NLRP3 or its mutants and Flag-TRIM31 were individually transfected into HEK293T cells. The cell lysates were immunoprecipitated with an anti-Flag antibody and then immunoblotted with the indicated antibodies. Similar results were obtained in three independent experiments.