Syk and Lyn kinases phosphorylate tyrosine residues in recombinant HGAL protein in vitro. (A) A schematic diagram of HGAL protein showing location of 6 tyrosines, a putative SH2 domain binding motif (YEN), and a first tyrosine (Y128) of modified immunoreceptor tyrosine-based activation motif (ITAM). Microcapillary reverse-phase high-performance liquid chromatography nanoelectrospray tandem mass spectrometry demonstrated that recombinant HGAL protein is phosphorylated on Y80, Y86, Y106Y107, and Y128 (in red) by Syk and Lyn kinase in vitro. Y148 (in black) was already phosphorylated before the addition of kinases. (B) Western blot of HGAL protein following incubation of Trx-HGAL recombinant protein with recombinant Lyn, Syk, or bovine serum albumin (BSA) in a kinase assay. Upward shift of the band in the presence of kinases suggests HGAL phosphorylation, eliminated by the addition of γ-phosphatase. (C-D) Recombinant Trx-HGAL was incubated with Syk kinase or Lyn kinase or bovine serum albumin in a kinase assay cocktail containing ³²P-adenosine triphosphate. Samples were loaded on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel, dried, and exposed to radiographic film. Data in panels B-D are representative of 3 independent experiments. kDa, kilodaltons.