Hck phosphorylates p73α in vivo and in vitro. (A) Whole cell lysates prepared from Cos1 cells transfected with HA-p73α (1.6 μg) with and without Hck (0.4 μg) expression constructs in the ratio of 4:1 were subjected to immunoblotting with pTyr, p73 and Hck antibodies. (B) Cos1 cells transfected with indicated expression constructs were subjected to immunoprecipitation with p73 antibody and immunoprecipitates analyzed for pTyr, p73 and Hck by western blotting. The amount of DNA was kept constant by addition of control vector pcDNA3. (C) Purified Hck protein (80 nM) was incubated with GST, GST-p73α and alone with γ32P-ATP for 30 minutes at 37°C in an in vitro kinase assay. The proteins were analyzed by SDS-PAGE and stained with commassie blue (left panel). The gel was then dried and phosphorylated proteins visualized by phosphor imaging (right panel). (D) Western blot showing the endogenous protein levels of p73 and Hck upon differentiation with DMSO in HL-60 cells. Tubulin expression was determined as a loading control. (E) Endogenous p73 gets phosphorylated on tyrosine upon activation of Hck. Western blot showing the phosphotyrosine content of cellular proteins and levels of endogenous p73 and Hck upon HgCl2 treatment in differentiated HL-60 cells (left panel). Lysates of diferentiated HL-60 cells treated with or without HgCl2 were subjected to immunoprecipitation with control (rabbit IgG) or p73 (rabbit polyclonal) antibody and western blotting performed with anti-phosphotyrosine antibody (right panel, upper portion) Immunoprecipitated p73 is shown in right panel, lower portion.