Fig. 6

S658 phosphorylation is required for NLRP3 inflammasome activation. (A) Heatmap demonstrated the differential phosphorylation peptides (fold change > 1) from 4D label-free quantitative phosphorylation proteomics. (B) Representative phosphorylation peptide Q3UZ39, annotated to NLRP3 protein. (C) The phosphorylation sites blocked by Psoralen across different species. BMDMs isolated from Nlrp3 KO mice were transfected with wtNLRP3, mutNLRP3-S658A plasmids, or mutNLRP3-S658D plasmids, then ELISA detected the production of IL-1β, IL-6, and TNF-α in the supernatant of BMDMs (D), Microglia (E), and Astrocytes (F). n = 6. (G) IL-1β and caspase-1 from SN and pro-IL-1β/pro-caspase-1 from Lysates were analyzed by immunoblotting. (H-I) Phos-tag SDS-PAGE and quantification of NLRP3 phosphorylation levels in Nlrp3 KO BMDMs transfected with wtNLRP3, mutNLRP3-S658A plasmids, or mutNLRP3-S658D plasmids. (J-K) Immunofluorescence staining and quantification of ASC (red) in Nlrp3 KO BMDMs transfected with wtNLRP3, mutNLRP3-S658A plasmids, or mutNLRP3-S658D plasmids. DAPI stains the nucleus (blue). The scale bar represents 5 μm. Data were analyzed by two-way ANOVA, followed by Tukey post-tests. ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001. ns: no significance