FIG. 2.
Endothelial p85 associates preferentially with Y1101 of the Tie2 kinase in vitro. Similar amounts of recombinant GST-Tie2 kinase fusion proteins, with or without specific mutations, were immobilized on glutathione-Sepharose, subjected to in vitro kinase reactions, and incubated with endothelial cell lysates. Kinase-associated proteins were separated by SDS–8 to 16% PAGE and analyzed by Western blotting with antibodies against Tie2, phosphotyrosine (PTyr), p85, and Grb2. An aliquot of crude endothelial cell lysate (lysate) was used as a comparison for p85 and Grb2, and glutathione-Sepharose alone, without recombinant kinase, was incubated with endothelial cell lysates as a negative control (beads).