Elm1 exhibits Gin4-dependent phosphorylation in vivo and Gin4 heavily autophosphorylates in vitro. (A) Analysis of immunoprecipitated Elm1-GFP from control (YEF9327, no GFP), WT (YEF10749, ELM1-GFP), and gin4∆ (YEF10750, gin4∆ ELM1-GFP) whole cell lysates separated via SDS-PAGE and immunoblotted with anti-GFP antibody (left) or Coomassie Blue stained (right). Yellow boxes indicate the gel area excised for mass spectrometry analysis. IP = immunoprecipitated, IB = immunoblotted. (B) Protein schematic of Elm1 with indicated domain boundaries labeled with amino acid positions. Green values indicate the position of a phosphorylated residue discovered via mass spectrometry only in the WT Elm1-GFP IP sample. Red values indicate the position of a phosphorylated residue found enriched in the gin4∆ Elm1-GFP IP mass spectrometry sample compared with that of the WT sample. Blue values indicate the position of a phosphorylated residue found enriched in the WT Elm1-GFP IP mass spectrometry sample compared to that of the gin4∆ sample. (C) Phosphorylated residues discovered in the 6xHis-SUMO-Gin4 protein after kinase reaction in the presence of GST-Elm1^KD. Asterisk (*) indicates phosphorylated residues believed to be Elm dependent from a previous study (Asano et al., 2006).