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. 2022 Nov 17;298(12):102717. doi: 10.1016/j.jbc.2022.102717

Figure 4.

Figure 4

LRR domain deletion mutants of NLRP3 show defects in NLRP3 self-association and oligomerization.A, HA-tagged mouse full-length NLRP3, LRR domain deletion mutant NLRP3 (1–686), or NLRP3 (1–720) was expressed alone or with indicated SFP-tagged protein in HEK 293T cells. Cell lysates were immunoprecipitated with an anti-Flag antibody and immunoblotted with indicated antibodies. B, mouse Nlrp3−/− macrophages were reconstituted with SFP-tagged wildtype or mutant NLRP3 and stimulated with PBS (mock), ATP, or nigericin after LPS priming. NLRP3 oligomerization was analyzed by blue native PAGE and immunoblotting with an anti-Flag antibody. Cell lysates were also analyzed by SDS–PAGE and immunoblotting with indicated antibodies. C, macrophage cell lysates were separated by the blue native PAGE, followed by a second dimension of SDS–PAGE and Western blot. Representative blots (n = 3). FL, full-length; LRR, leucine-rich repeat; SFP, S-tag, Flag, and streptavidin-binding tag.