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. Author manuscript; available in PMC: 2021 Apr 4.
Published in final edited form as: Exp Hematol. 2020 Apr 4;84:29–44. doi: 10.1016/j.exphem.2020.03.003

Figure 1. Proteomic profiling of EPO/EPOR/JAK2 regulated phospho-PTM targets.

Figure 1.

(A-F) Phospho-PTM workflow, and characterization of EPO-dependent human erythroid progenitor UT7epo-E cells. A: In the outlined phospho-PTM proteomic workflow, steps employed to profile EPO-regulated p-Y or p-TPP motif modified target proteins in UT7epo-E cells are defined. B: Analyses of mutations in 18 myeloproliferative driver genes were assessed, with no such mutations detected. C: Profiling of erythroid vs. megakaryocytic marker transcripts in UT7epo-E cells indicated a predominant erythroid phenotype. D: HBA globin levels in UT7epo-E cells (via western blotting) approximate levels in primary basophilic erythroblasts (western blot, right panel). E,F: UT7epo-E cells retain sharp EPO dose- dependency for growth and survival (E), and dynamic cell surface EPOR expression (F) (flow cytometry assays). (G,H) Validation data for known EPO- modulated phospho-PTM targets – G: To illustrate insight generated via this affinity LC-MS/MS approach, PTM-modulated signal transduction factors known to associate with EPO/EPOR/JAK2 complexes are first considered. Data illustrated for ten validation targets include: The fold-modulation of specific phospho-PTMs (“EPO, fold-modulation” column); parent target protein identities (“protein target” column); the use of trypsin (t) or Glu-C (g) to generate peptides (“study” column); EPO-regulated phospho-residues within the parent protein sequence (“site(s)” column); LC-MS/MS defined sequences of the EPO-regulated phospho-peptide (“peptide” column); and identifiers for parent proteins (“protein description” column). H: For comparison, western blot signals for select known EPO- regulated phospho-PTM targets are also shown including p-Y1007/p-Y1008-JAK2, p-Y570-JAK2, p-Y132/p-Y141-RHEX, and p-Y986/p-Y987 INPPL1.