Evidence that Y78 and Y112 of TpoR are phosphorylated in TpoRW515A cells selected for autonomous growth. Phosphopeptides were prepared using PhosphoScan Kit (Cell Signaling Technology) after lysis of Ba/F3 TpoRW515A cells in urea buffer. Trypsin-digested lysates were immunoaffinity purified with pY-100 antibody, concentrated on reverse-phase microtips, and analyzed by mass spectrometry. (A) Mass spectrum representing phosphorylation of Y78 of TpoR (Y582, full murine TpoR numbering). (B) Mass spectrum representing phosphorylation of Y112 of TpoR (Y616, full murine TpoR numbering). Oxidized methionine is represented by M# and tyrosine phosphorylation, by Y*. Phosphorylated residues are denoted as y. (C) Schematic representation of TpoR with the location of the W515A mutation in the TpoR cytosolic juxtamembrane domain (red region) and the Y78 and Y112 in the cytosolic domain of the receptor.