Figure 4
Interaction of Pyk2 and FAK with ASC.
(A) Immunoblot analysis of FAK and ASC in immunoprecipitants prepared with anti-FAK and the corresponding normal rabbit IgG from extracts of HEK293T cells obtained at 48 h after transfection of a vector encoding ASC-Flag. The western blot is a representative of three independent experiments. (B) Immunoblot analysis of Pyk2-Flag and ASC in immunoprecipitants prepared with anti-Flag matrix and the corresponding normal mouse IgG, from HEK293T cell extracts obtained 48 h after co-transfection of vectors encoding Pyk2-Flag and ASC. (C–F) The localization of FAK and Pyk2 in ASC-mCherry-expressing THP-1 cells stimulated for 1 h with nigericin (C,D) and for 4 h with poly(dAdT) (E,F) was visualized by immunostaining with an anti-FAK or anti-Pyk2 antibody (green). ASC is shown in red, while nuclei are blue. Scale bars, 10 μm. (G–J) In situ PLA of PMA-differentiated THP-1 cells stimulated for 1 h with nigericin (G,H) and for 4 h with poly(dAdT) (I,J). (G,I) Complexes of phosphorylated FAK with ASC (p-FAK + ASC, green). (H,J) Complexes of phosphorylated Pyk2 with ASC (p-Pyk2 + ASC, green). ASC is shown in red, while nuclei are blue. The results were quantified using an IN Cell Analyzer, and are presented relative to the value obtained from unstimulated control cells. Scale bars, 10 μm.
