Figure 3

The SH2 domain is important for Grb14 tyrosine phosphorylation. A) HEK 293T cells were transfected with Tie2, either WT or the kinase inactive K853A mutant. Lysates were incubated with immobilized GST alone, GST-SH2 domains from Grb2 (control) or Grb14 (as indicated). Resulting complexes were resolved by SDS-PAGE and immunoblotted with anti-Tie2 antibodies. B) HEK 293T cells were co-transfected with Tie2 (WT or kinase inactive) and FLAG tagged Grb14 (WT or the FLVRS mutant). Lysates subjected to immunoprecipitation by either Tie2 or FLAG antibodies and resolved by SDS-PAGE. Western blotting with indicated antibodies was then carried out.