Figure 4: TRIM31 promotes K48-linked polyubiquitination of NLRP3. | Nature Communications

Figure 4: TRIM31 promotes K48-linked polyubiquitination of NLRP3.

From: The E3 ubiquitin ligase TRIM31 attenuates NLRP3 inflammasome activation by promoting proteasomal degradation of NLRP3

Figure 4

(a) Immunoblot analysis of lysates from HEK293T cells transfected with HA-tagged ubiquitin (HA-Ub), Myc-NLRP3 and TRIM31 WT or C16AC36A, followed by IP with anti-Myc, probed with anti-HA or K48-Ub. (b) Immunoblot analysis of lysates from HEK293T cells transfected with HA-tagged K48-linked ubiquitin (K48-Ub) or HA-tagged K63-linked ubiquitin (K63-Ub), Myc-NLRP3 and TRIM31, followed by IP with anti-Myc, probed with anti-HA. (c) Immunoblot analysis of lysates from TRIM31+/+ or TRIM31−/− mouse peritoneal macrophages, followed by IP with anti-NLRP3, probed with anti-Ub, K48-Ub or K63-Ub. (d) In vitro ubiquitination assay was performed in the presence of Ub (wt, K48 or K63), E1, E2-UbcH5A, NLRP3 and TRIM31 (wt or ΔRING mutant). The ubiquitination of NLRP3 was examined with NLRP3 antibody. (e) Immunoblot analysis of extracts from HEK293T cells transfected with Myc-NLRP3 and Flag-tagged TRIM31 or its mutants. (f) Immunoblot analysis of extracts from THP-1 cells transfected with TRIM31 wild type (WT) or TRIM31 C16AC36A. (g) Immunoblot analysis of extracts from THP-1 cells transfected with TRIM31 WT or TRIM31 ΔRING then stimulated with LPS for 4 h. Similar results were obtained in three independent experiments.

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