Fig. 2: PYK2 interacts with IRF5 and controls IRF5 activation.

a Endogenous co-immunoprecipitation in RAW264.7 macrophages. Cells were stimulated with LPS for 10 min and immunoprecipitated with IRF5, PYK2 or an isotype control antibody. Immunoprecipitates were eluted from IP beads and proteins present in cell lysates (5% inputs) and eluates were detected by immunoblotting with antibodies against IRF5 or PYK2. b Immunoblot of LPS-induced PYK2 tyrosine phosphorylation. Blots were probed with antibodies specific for PYK2 phosphorylated on Tyr-402, total PYK2 and GAPDH. Representative blots from three independent experiments are shown for (a, b). c TNF-luc reporter activity in the absence or presence of ectopically expressed IRF5 in wild type and PYK2 KO RAW264.7 cells stimulated with LPS 6 h or left untreated. AU arbitrary units of luminescence. d IRF5 and pol II binding to Il6 and Il1a gene promoter in resting or LPS-treated (2 h, 500 ng/ml) wild type or PYK2 KO RAW264.7 cells as measured by the chromatin immunoprecipitation (ChIP) method. A non-specific IgG antibody was used as a negative control for ChIP. e Il6 and Il1a mRNA induction in wild type, PYK2 KO or IRF5 KO RAW264.7 cells stimulated with LPS (500 ng/ml) for 0, 2, or 4 h. Gene expression was measured by qPCR. All values in (c–f) are shown as mean values ± SEM from n = 3 independent experiments. Statistical significance was calculated with two-way ANOVA with Sidak’s correction *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. f Gene expression levels in wild type, PYK2 KO or IRF5 KO HoxB8 macrophages stimulated with LPS (100 ng/ml) 2 h. Gene expression was measured by qPCR. Values shown as mean values ± SEM from n = 3 experiments. Comparison by two-way ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001). Source data are provided as a Source Data file.