Figure 2. GSK3β phosphorylates ETS1 protein. (A) The ETS1 protein is characterized by the presence of conserved GSK3β phosphorylation motifs in different species (human, mouse, rat, and chicken). Two potential motifs (highlighted in red and green letters) underwent mutagenesis and were termed ETS1-3A (red letters) and ETS1-2A (green letters). (B) In vitro kinase assay. ETS1, ETS1-3A, and ETS1-2A were transfected into 293 cells and subsequently purified using a halo resin. After incubation with GSK3β and ATP, phosphorylated ETS1 was detected using anti-threonine/serine antibody. (C) Phosphorylated peptides of the ETS1 protein were subjected to an in vitro kinase reaction either with or without GSK3β following enrichment with TiO2 beads. Subsequently, an LC-MS/MS analysis was performed. The ion chromatograms of the peptide potentially targeted by GSK3β (LTQSWSSQSSFNSLQR at amino acids 264-279 of the ETS1 protein) with single (968.435±0.005 m/z, upper panel, white triangle) and double (1008.415±0.005 m/z, lower panel, black and white arrows) phosphorylation sites were extracted. Two double phosphorylation events were identified – with a relatively marked difference with and without GSK3β treatment.