Lyn kinase mediates IL-6–induced tyrosine phosphorylation of the HGAL protein. (A) VAL and HeLa-HGAL cells were treated with IL-6 for up to 15 minutes. The whole-cell lysates were extracted, separated by SDS-PAGE, and detected with the indicated antibodies. (B) VAL cells were transfected with either siRNA for Lyn or scrambled control, respectively. At 48 hours after transfection, the cells were stimulated with IL-6 (10 ng/mL) for 10 minutes and cellular lysates were extracted, immunoprecipitated with anti-HGAL antibody, separated by SDS-PAGE, and immunoblotted with antiphosphotyrosine or anti-HGAL antibodies. (C) HeLa-HGAL cells were transiently transfected with plasmids encoding for Lyn-Y508F and Lyn-K275D. At 48 hours after ransfection, whole-cell lysates were prepared, immunoprecipitated with anti-HGAL antibody, separated by SDS-PAGE, and immunoblotted with antiphosphotyrosine or anti-HGAL antibodies. Unstimulated and IL-6–stimulated HeLa-HGAL cells served as controls. In panels A-C, representative blots of 2 independent experiments are shown.