Peptide pull-downs were performed on NB1 cell lysate [prepared using lysis buffer containing 50 mM tris (pH 8.5), 150 mM NaCl, 10 mM potassium chloride, 0.1% Triton X-100, and 0.5 mM dithiothreitol (DTT) with the addition of 5 mM β-glycerophosphate, 5 mM sodium fluoride, 1 mM sodium orthovanadate, and 1 cOmplete inhibitor cocktail tablet per 10 ml (Roche)], from DMSO- and LDK378-treated cells using biotinylated peptides; phosphotyrosine-containing peptides or the corresponding nonphosphorylated version (fig. S1C). Peptides included the following phosphotyrosines (±6 amino acids flanking the indicated phosphotyrosine residues) from ALK (Tyr1078, Tyr1092, Tyr1096, Tyr1131, Tyr1278, Tyr1283, Tyr1283, Tyr1359, Tyr1507, Tyr1584, and Tyr1604) and from IRS2 (Tyr675, Tyr742, and Tyr978), including the nonphosphorylated counter peptide synthesized by Intavis and coupled to Sepharose-streptavidin beads (GE Healthcare) in a buffer containing 50 mM tris (pH 8.5), 150 mM NaCl, and 0.1% NP-40. Pull-downs (1 mg of cell lysate per pull-down), elution, and on-bead trypsin digestion were carried out in 96-well multiscreen filter plates essentially as described by Eberl et al. (72).

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