Spry1 is strongly and rapidly PY-phosphorylated on EPOR activation. (A) Primary bone marrow–derived erythroid progenitors do not express substantial levels of FGF receptors. In purified erythroid stage E1, E2, and E3 cells, transcriptome and RT-PCR analyses were applied to assess FGFR-1, -3, and -4 levels. (B) The LC-MS/MS approach used to discover novel EPOR PY-modulated signal transduction factors (including Spry1) in erythromegakaryocytic UT7epo cells is outlined. (C) Multifold EPO/EPOR stimulation of Spry1 phosphorylation at PY-Y53. Data shown illustrate results for duplicate LC-MS/MS analyses. (D) Verification of LC-MS/MS sensitivity and specificity based on EPO/EPOR regulation of JAK2 PY phosphorylation (PY sites Y-1007, Y-1008, Y-221, Y570, and Y-813) and GAB2 PY phosphorylation (PY sites Y643, Y614, and Y476).