Figure 5
ASC Tyr146 is phosphorylated by Pyk2, and this is essential for ASC oligomerization.
(A,B) In situ PLA of phosphorylated tyrosine-ASC complexes in PMA-differentiated THP-1 cells stimulated for 1 h by nigericin in the presence or absence of PF-431396. (A) Complexes of phosphorylated tyrosine with ASC (p-Tyr + ASC; green). ASC is shown in red, while nuclei are blue. The results were quantified with an IN Cell Analyzer, and are presented relative to the value obtained from unstimulated control cells. Scale bars, 10 μm. (B) An in vitro kinase assay of FAK and Pyk2 was performed by incubating recombinant His-ASC with His-FAK or His-Pyk2, as indicated. The protein amounts were assessed by immunoblotting with anti-FAK, anti-Pyk2, and anti-ASC antibodies. (C) An in vitro kinase assay of Pyk2 was performed by incubating recombinant His-Pyk2 with wild-type or mutant (Y146F) His-ASC. The protein amounts were assessed by immunoblotting with anti-Pyk2 and anti-ASC antibodies. (D) Analysis of ASC oligomerization in reconstituted HEK293T cells 48 h after co-transfection of empty vector or Flag-NLRP3-encoding vectors plus wild-type or mutant (Y146F) ASC-Flag-encoding vectors. The western blot is a representative of three independent experiments. (E) Analysis of ASC oligomerization in reconstituted HEK293T cells 48 h after co-transfection of vectors encoding Flag-NLRP3 or mutant (Y146F) ASC-Flag along with increasing amount of vector encoding wild-type ASC, and immunoblotting was performed as indicated using anti-ASC (top panel) and anti-Flag (middle panel) antibodies. The western blot is a representative of three independent experiments. Relative amounts of mutant ASC (Y146F) homodimers, wild-type/mutant ASC heterodimers, and wild-type ASC homodimers from this representative blot are shown at the bottom panel. Symbol: *non-specific signal.
