Egr-1, an immediate-early gene product and master regulator was originally described as a phosphoprotein following its discovery in the 1980s. However specific residue(s) phosphorylated in Egr-1 remain elusive. Here we phosphorylated recombinant Egr-1 in vitro with ERK1 prior to mass spectrometry, which identified phosphorylation of Ser(12) and Ser(26) with the latter similar to 12 times more abundant than Ser(12). Phosphorylation of wild-type recombinant Egr-1 (as compared with Ser(26)>Ala(26) mutant Egr-1) revealed that Ser(26) accounts for the majority of phosphorylation of Egr-1 by ERK1. N-FGSFPH(pS)PTMDNYC-C was used as an antigen to generate mouse monoclonal antibodies (pS26 MAb). pS26 MAb recognised ERK1-phosphorylated Egr-1 but not Egr-1 bearing a point mutation at Ser(26). pS26 MAb recognised inducible similar to 75 kDa and 100 kDa species in nuclear extracts of cells exposed to FGF-2. Peptide blocking revealed both inducible species were phosphosite-specific. lmmunoprecipitation of nuclear extracts of cells exposed to FGF-2 with pS26 MAb followed by SDS-PAGE and mass spectrometry identified Egr-1 sequences corresponding to the similar to 75 kDa species but not similar to 100 kDa species. This study identifies a specific amino acid phosphorylated in endogenous Egr-1. (C) 2019 Published by Elsevier Inc.