Cell lysates for immunoprecipitation were prepared as described previously (29). Cell lysates (1 to 2 mg) were incubated overnight at 4°C with anti-ALK antibody (Cell Signaling Technology) or anti-IRS2 antibody (Abcam), with subsequent binding to protein G–Sepharose for 1 hour. After five washes with lysis buffer, the bound proteins were eluted by boiling in SDS sample buffer and resolved by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by Western blotting.

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