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. Author manuscript; available in PMC: 2022 Apr 26.
Published in final edited form as: Sci Signal. 2021 Oct 26;14(706):eabe3410. doi: 10.1126/scisignal.abe3410

Fig. 3. Lyn suppresses the activation of NLRP3 inflammasome.

Fig. 3.

(A) WT BMDMs were transfected with Ctr siRNA or Lyn siRNA by lipofectamine 2000, primed with LPS for 4 hours, then stimulated with ATP for 5 min, and lysed in RIPA buffer. The cell lysates were immunoprecipitated with anti-NLRP3 and blotted with antibodies to pTyr and ubiquitin. (B) WT BMDMs treated with siRNA and primed as in A, and then stimulated with ATP for 30 min. The expression of NLRP3 was determined by immunoblot analysis. (C) Immunoblot analysis for caspase-1 (Casp-1) p10 and IL-1β p17 pre-processed “pro” forms in whole cell extracts (CL) from (B) and their cleaved form in the same supernatants (sup). IL-1β production in supernatants under the same conditions was measured by ELISA. (D) BMDMs from WT and Lyn–/– mice, primed with LPS, and stimulated with ATP. The cell lysates were immunoprecipitated with antibody to NLRP3 and blotted with antibodies to pTyr and ubiquitin. (E) Immunoblot analysis for NLRP3 in cell lysates from LPS-primed WT and Lyn–/– BMDMs stimulated with ATP. (F) Immunoblot analysis for caspase-1 (Casp-1) p10 and IL-1β p17 pre-processed “pro” forms in whole cell extracts (CL) from (E) and their cleaved form in the same supernatants (sup). IL-1β production in supernatants under the same conditions was measured by ELISA. (G) ELISA detection of IL-1β in the supernatants from LPS-primed BMDMs from WT and Lyn–/– mice treated as indicated with ATP or ATP with Ac-YVAD-cmk, nigericin, MSU, poly(dA:dT), and flagellin. (H) LDH assay of LPS-primed BMDMs and stimulated with ATP in the presence of VX765 or VX765 plus MG132. (I) Immunoblot analysis for NLRP3 in cell lysates (both solube and insoluble fractions) from LPS-primed WT BMDMs treated as in (H). The purity of soluble and insoluble fractions was determined by immunoblotting with tubulin and vimentin, respectively. The data are from a representative one of three (A to F), two (G and H), or four (I) independent experiments. Data are mean ± SD for IL-1β in (C, G, and H). *P< 0.05; **P< 0.01, student t test.