Fig. 1.

Schematic overview of the SILAC approach used to identify IR-A mediators after IGF-II or insulin stimulation. Cells were grown in three different arginine-stable isotope-labeled media. Cells grown in natural arginine were stimulated with vehicle alone. Cells grown in l-13C6-Arg-containing medium were treated with IGF-II, and cells grown in l-13C615N4-Arg-containing medium were treated with insulin. Stimulation was carried out with 10 nm ligand for 1 min. Equal amounts of proteins were mixed with antiphosphotyrosine antibodies (anti-pY) and the immunoprecipitated (IP) fraction was resolved by SDS-PAGE. The gel was excised into bands, and each band was digested with trypsin. Each fraction from the in-gel digestion was analyzed by nano LC-MSMS, using a QSTAR XL hybrid MS instrument or an LTQ Orbitrap XL.