FIG. 5.

Recombinant Grb2 and PI3K-C2β associate in vitro. Grb2-GST fusion protein (1 μg) was added to Triton lysis buffer (500 μl) in the absence or presence of EE epitope-tagged PI3K-C2β (1 μg) and either glutathione-Sepharose beads or anti-EE tag antibody (Ab) and protein A-Sepharose. After incubation at 4°C for 4 h, beads were isolated by centrifugation and washed. Associated proteins were extracted, fractionated by SDS-PAGE, and Western blotted with either anti-PI3K-C2β antibody (upper panel) or anti-Grb2 antibody (lower panel) and visualized with ECL.