Figure 4.

S5 phosphorylation prevents Trim31-mediated ubiquitination of NLRP3 at K496 site.

(A) Immunoblots of the whole cell lysates of control-siRNA-, AKT1/2-siRNA-, or/and Trim31-siRNA-transfected LPS-primed THP-1 cells. The endogenous NLRP3 protein levels were normalized to control-siRNA transfected untreated sample (showed in fold).

(B) Quantification of the Trim31 mRNA level in control-siRNA- or Trim31-siRNA-treated THP-1 cells. Control-siRNA or Trim31-siRNA was transfected into THP-1 cells for 48 hrs, and the trim31 mRNA level was tested from real-time PCR. Experiments were repeated three times, and bars represent the means ± SEM. *p<0.05, as determined by student’s t test.

(C) Immunoblots of the whole cell lysates of the HEK293T cells transfected with Flag-NLRP3 (WT or S5A mutant), and Flag-Trim31 (WT or ΔRing mutant). Expressions of transfected NLRP3 protein level were normalized to vector-transfected groups, respectively.

(D) Immunoblots showing the ubiquitination of NLRP3. AU1-Trim31 and HA-Ubiquitin were co-transfected into HEK293T cells with Flag-NLRP3 WT or S5A mutant, followed by treatment of MK2206 (100 nM, 6 hrs.) and MG132 (20 μM, 4 hrs). The whole cell lysates were immunoprecipitated with anti-NLRP3 and immunoblotted with indicated antibodies.

(E) Uniprot alignment of NLRP3 orthologs surrounding Lysine 496 site.

(F) Immunoblots of the whole cell lysates of the HEK293T cells transfected with Flag-NLRP3 (WT or K496R mutant), and Flag-Trim31 (WT or ΔRing mutant). Expressions of transfected NLRP3 protein level were normalized to vector-transfected groups, respectively.

(G) Immunoblots showing the ubiquitination of NLRP3. AU1-Trim31 was co-transfected into HEK293T cells with Flag-NLRP3 WT or K496R mutants, followed by treatment of MG132 (20 μM, 4 hrs.). The whole cell lysates were immunoprecipitated with anti-Flag and immunoblotted with indicated antibodies.

(H) Immunoblots of the NLRP3 inflammasome reconstitution in HEK293T cells. To reconstitute the NLRP3 inflammasome, Flag-tagged ASC, Flag-tagged pro-caspase-1, and AU1-tagged Trim31 were transfected with Flag-NLRP3 WT or K496R mutant in HEK293T cells, and then respectively treated with Nigericin (5 μM, 45 min) before collection. Immunoblots shown are representative of at least 3 independent experiments and band quantification is shown for the representative blot.