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. Author manuscript; available in PMC: 2016 Jun 1.
Published in final edited form as: Nat Struct Mol Biol. 2015 Nov 9;22(12):983–990. doi: 10.1038/nsmb.3117

Figure 3.

Figure 3

Src primes Shc1 for EGFR phosphorylation by phosphorylating Tyr240. (a) Peptide library results for Src. Representative image from 3 independent experiments is shown. Quantification is provided in Supplementary Table 2. (b) Src kinase assays with synthetic peptides corresponding to the sequence surrounding Tyr239 of Shc1 (PDHQYYNDAKKK = Y-Y; PDHQYpYNDAKKK = Y-pY; PDHQpYYNDAKKK = pY-Y). Error bars, s.d. (n=3 technical replicates) (c) HPLC analysis at of a Src kinase assay with the Shc1 Y-Y peptide at multiple time points. (d) Mass spectrometry based analysis of the 120 min peak in panel C. The LC-MS/MS spectrum for the singly phosphorylated peptide PDHQYpYNDAKKK is shown. (e) Percentage of peptide spectra detected by LC-MS/MS phosphorylated at Tyr239 or Tyr240. (f) HPLC analysis of an in vitro kinase assay containing Src, EGFR and the Y-Y Shc1 peptide at multiple time points.