Fig. 1. NLRP3 tyrosine phosphorylation is required for its ubiquitination.

(A) ELISA detection of IL-1β in the supernatants from LPS-primed BMDMs treated as indicated with ATP（2.5 mM, 30 min), nigericin (20 μM, 3 hours), MSU (200 μg/ml, 4 hours), silica (500 μg/ml, 6 hours), poly(dA:dT) (1 μg/10⁶ cells, 6 hours), or flagellin (6.25 μg/10⁶ cells, 4 hours), each either alone or in the presence of Src-I1 (Src-I; 44 nM, 1 hour) or BAY 61–3606 (BAY-I; 10 nM, 1 hour). Below, Western blotting for caspase-1 (Casp-1) p10 and IL-1β p17 in the same supernatants (sup) and their pre-processed “pro” forms in whole cell extracts (CL). (B) WT BMDMs were primed with LPS and stimulated with ATP or nigericin in the presence of Src-I1 or BAY 61–3606, then lysed in RIPA buffer. The cell lysates were either immunoprecipitated with antibody to NLRP3 and blotted with antibodies to pTyr and ubiquitin, or they were immunoprecipitated with antibody to pTyr and blotted with antibody to NLRP3. (C) Western blotting for ASC in cell lysates (either cross-linked with DSS or not) from LPS-primed WT BMDMs pretreated with Src-I1 or BAY 61–3606 and stimulated with ATP. Input control (CL) is from pre-crosslinked aliquots. The data are from a representative one of three (A and B) or 4 (C) independent experiments. *P< 0.05, student t test.