Fig. 1

GEF-H1 mediates IRF5 phosphorylation during MDP recognition. a Immunoblot analysis of the activation of IRF5 with antibodies detecting phosphorylated (p-) or total IRF5 in whole-cell lysate of bone marrow derived macrophages (BMDMs) from WT, Arhgef2^−/− and Nod2^−/− mice after stimulation with N-glycolyl-MDP (5 μg ml⁻¹) for 1 h and 4 h. β-actin used as loading control. b Nod2 mRNA expression analysis by qRT-PCR from WT, Arhgef2^−/− and Nod2^−/− derived BMDMs and genotyping PCR for targeted or wildtype Nod2 allele in genomic DNA from Nod2^−/−, WT and Arhgef2^−/− mice. c Immunoblot analysis of IRF5 expression in nuclear extracts from BMDMs from WT, Arhgef2^−/− and Nod2^−/− mice after stimulation with N-glycolyl-MDP with band intensities quantified using densitometry and Image J software. d Immunoblot analysis of GEF-H1 expression after IRF5 pull down in BMDMs from WT and Arhgef2^−/− mice in presence or absence of N-glycolyl-MDP. e Immunoprecipitation of GEF-H1 from HEK293T cells that were transfected with IRF5, GEF-H1 and/or RIPK2 encoding plasmids. f Western blot analysis of the expression and phosphorylation of IRF5 in BMDMs from WT and Ripk2^−/− mice that were stimulated with N-glycolyl-MDP for 1 h and 4 h. g Immunoblot analysis of the expression and phosphorylation of IKKα/β and IKKε in BMDMs from WT and Ripk2^−/− mice after stimulation with N-glycolyl-MDP for 1 h and 4 h. h Immunoprecipitation of GEF-H1 with specific ABs from HEK293T cells that were transfected with GEF-H1-Flag, IKKε-Flag, ΙΚΚβ-HA, and NOD2-Flag expression constructs. Anti-GEF-H1, anti-IKKε, and anti-IKKβ were used to detect proteins by western blotting and NOD2 was detected with anti-Flag. Source data are provided as a Source Data file