Fig. 4: FGF4–ERK1/2 signalling inhibits EPHA2 activation and rewires EPH–EFN expression.

a Diagram of potential phosphorylation sites within the EPHA2 S898 motif. Mass spectrometry analysis detects phosphorylation of at least three sites in EPHA2 immunoprecipitated from FGF4-stimulated Fgf4^−/− mESCs (see Supplementary Table 1). b Fgf4^−/− mESCs were treated with 10 μM of the indicated inhibitors for 1 h, and stimulated with FGF4 for 10 min. EPHA2 pS898, EPHA2 and AKT pS473 levels were determined by immunoblotting. c Epha2^−/− mESCs were transfected with either wild type or 5E EPHA2 constructs, and stimulated with 1 μg/ml clustered EFNA1 for 15 min. EPHA2 was immunoprecipitated, and pTyr and EPHA2 levels determined by immunoblotting and quantified. Data show mean ± SD (n = 4). d EPHA2 was immunoprecipitated from EPHA2 WT knock-in (KI) or 5A KI cell lines and pTyr and EPHA2 levels determined by immunoblotting (upper panel). Relative pTyr/EPHA2 signal was quantified (lower panel). Data show mean ± SD (n = 4). e Phase-contrast images of EPHA2 WT KI or 5A KI mESC lines; scale bar = 100 µM. f EPHA2 WT KI or 5A KI cell lines were cultured in LIF/FBS medium for 48 h, and KLF4, NANOG, DNMT3B, OCT4 ppERK1/2 and ERK1/2 levels determined by immunoblotting. g Epha2^+/+ mESCs were differentiated as embryoid bodies for 10 days, and EPH receptor expression determined by qRT-PCR at the indicated time points. Box-and-whisker plots show median, first and third quartiles, and maximum and minimum values of four technical replicates (n = 4). h Epha2^+/+ mESCs were differentiated as embryoid bodies for 10 days, and EFN ligand expression determined by qRT-PCR at the indicated time points. Box-and-whisker plots show median, first and third quartiles, and maximum and minimum values of four technical replicates (n = 4). Epha2 (i) and Efna1 (j) mRNA expression in 2i mESCs undergoing differentiation in N2B27 was determined by qRT-PCR analysis at the indicated time points. Box-and-whisker plots show median, first and third quartiles, and maximum and minimum values of two technical and three biological replicates (n = 3). Source data are provided as a Source Data file.