Figure 2

Y1100 and Y1106 on Tie2 are important for Grb14 tyrosine phosphorylation. HEK293T cells were transfected with Grb14 alone or in combination with wild type Tie2, the kinase deficient Tie2 (K853A) or one of the Tie2 tyrosine mutants (Y1100F, Y1106F, Y1111F, Y1100/1106F). A) Lysates (100 μg) from these cells were immunoprecipitated using an antibody specific for Grb14, run out on an SDS PAGE gel and blotted for phosphotyrosine (top panel). Cell lysates (20 μg) were also run out on an SDS PAGE gel and subjected to Western blotting using an anti-Grb14 antibody as a control (bottom panel). B) Lysates were immunoprecipitated with an anti-Tie2 antibody and then either subjected to Western Blot using anti-Tie2 antibodies (top panel) or incubated in a kinase reaction with set amounts of glutathione-Sepharose purified GST-Dok-P^ΔPH and ³²P-labeled ATP (bottom panel).