Fig. 3.

Endogenous Ack forms a stable complex with the adaptor protein Nck. (A) Her14 cells were starved overnight in DMEM with 0.1% calf serum and then either mock-stimulated or stimulated with EGF (E) for 5 min. Precleared lysates (≈2 mg) were incubated with anti-Ack (Ab 729) or anti-Nck antibodies previously crosslinked to protein A Sepharose beads. Immunocomplexes were analyzed with anti-Ack (Ab 730) or anti-Nck antibodies. (B) 293 cells were transfected with expression vectors for Nck and wild-type or mutant Ack as shown. (Left) Precleared lysates (250 μg) were immunoprecipitated with anti-Nck antibodies and analyzed by immunoblotting with anti-Ack (Ab 880) or anti-Nck antibodies. (Right) Whole-cell lysates (WCL; 30 μg) were directly analyzed by immunoblotting with anti-Ack (Ab 880) or anti-Nck antibodies.