For each experiment, cells from three SILAC conditions (fig. S1C) were lysed in immunoprecipitation lysis buffer at 4°C. Proteins were precipitated overnight at −20°C in fourfold excess of ice-cold acetone. The acetone-precipitated proteins were solubilized in denaturation buffer [10 mM Hepes (pH 8.0), 6 M urea, and 2 M thiourea], and 5 mg of protein from each SILAC condition was mixed at a 1:1:1 ratio (total 15 mg per SILAC mix) and reduced with 1 mM DTT followed by alkylation with 5.5 mM chloroacetamide (CAA). Proteins were subjected to Lys-C digestion for 3 hours (Wako) and then, after a fourfold dilution using 50 mM tris (pH 8.0), digested with trypsin (Sigma-Aldrich) overnight at room temperature. The peptide mixtures were desalted and concentrated on C18 Sep Pak Cartridges (Waters) and eluted with 50% acetonitrile (ACN). Eluted peptide mixtures were dried almost to completeness in a SpeedVac and dissolved in Mops buffer [50 mM Mops (pH7.2), 10 mM sodium phosphate, and 50 mM NaCl] and subjected to anti-phosphotyrosine immunoprecipitation using a mixture of anti-phosphotyrosine antibodies (P-Tyr-100 and P-Tyr-1000; PTMScan kit, Cell Signaling Technology). Immunoaffinity beads were washed with increasing salt concentration (50 mM NaCl and 150 mM NaCl), and phosphopeptides were eluted with 0.1% trifluoroacetic acid (TFA) before loading onto a C18 StageTip. The unbound fraction from the anti-phosphotyrosine immunoprecipitation was acidified with TFA, desalted using a C18 Sep Pak Cartridge (Waters), and eluted with 50% ACN and 0.1% TFA. Peptide mixtures were adjusted to 80% ACN and 6% TFA, and phosphopeptides were further enriched by two sequential rounds of titansphere chromatography as previously described (73).

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