Fig. 1: Small molecule library screen and in vitro validation identifies PYK2 as a putative IRF5 kinase.

a The screening workflow showing the initial large screening in RAW264.7 cells, and the subsequent screens in RAW264.7 and 293 TLR4 cells. The top inhibitors were shortlisted based on their efficacy towards IRF5 reporter and low toxicity. Based on the known activities of these molecules against 221 kinases in the PKIS set, 34 kinases affected by the top inhibitors were shortlisted. The underlined kinases were previously proposed to target IRF5. b Impact of the kinases on the IRF5 reporter activity. Luciferase activities were measured in cells co-expressing IRF5 (or empty plasmid control, pBent2), TNF-luc reporter and one of the shortlisted kinases. Reporter activity was calculated as firefly luciferase activity normalised against constitutively expressed Renilla luciferase units and is shown as compared to the values in cells not expressing any kinase. AU arbitrary units of luminescence. Data presented as mean values ± SD for n = 3 independent experiments. c Binding of IRF5 to the shortlisted kinases. Myc-tagged kinases and HA-tagged IRF5 were co-expressed in 293 ET cells. Cell lysates were subjected to immunoprecipitation (IP) using anti-myc antibody and levels of kinases and IRF5 in the IP eluates and proteins were determined by western blot. Asterisks indicates the expected molecular weight. d In vitro kinase assays of 293 ET cells co-transfected with HA-IRF5 and myc- or flag-tagged kinases. Proteins in the pull-downs and lysates were detected by Western blotting using antibodies against HA- (IRF5) and myc- and FLAG- (kinases). e Schematic of IRF5 truncation mutants. f Schematic of PYK2 truncation mutants. g, h HEKTLR4 cells were co-transfected with Myc-PYK2 and Strep-tagged IRF5 truncation mutants and subjected to co-immunoprecipitation and western blot analysis. Representative blots from three independent experiments are shown. Source data are provided as a Source Data file.