Figure 4

Tyrosine phosphorylation of Grb14 in endothelial cells. A) Lysates from C166, EOMA, SVR, SVEC and EAhy92.6 endothelial cell lines as well as an HEK293 cell line that stably expresses the full-length Tie2 receptor (HEK293Tie2) were immunoprecipitated and then subjected to Western Blotting using an antibody specific for Grb14. Cell lysate from 293T cells overexpressing Grb14 was also run as a positive control. B) Human umbilical endothelial cells (HUVEC) were either left untreated or stimulated with the soluble and potent variant of the Tie2 ligand, cartilage oligomeric matrix protein (COMP) Ang1. Cell lysates were immunoprecipitated with anti-Grb14 and subjected to SDS PAGE and Western blotted for phosphotyrosine with 4G10 antibody. The positive control lane was protein sample derived from Grb14/Tie2Y1111 transfected 293T cells