Altered Epo receptor signaling in erythroid Lyn+/up cells compared with Lyn+/+ cells. (A) Altered phosphotyrosine dynamics during Epo induction and elevated EKLF, BclXL, CIS, SOCS1, and SOCS3 in J2-Lyn+/up cells. Time course of Epo-induced total phospho-tyrosine (pY) changes in J2-WT and J2-Lyn+/up cell lines. Prominent changes in phospho-proteins between the cell lines are indicated (arrows, and approximate kilodaltons). (B) Altered Lyn and proximal Epo receptor signaling dynamics during Epo induction in J2-Lyn+/up cells. Immunoblot analysis of J2-WT and J2-Lyn+/up cell lines for the signaling molecules indicated before and after 10 and 30 minutes of Epo stimulation. Total cell lysates were used for analysis except for pEpo receptor, where immunoprecipitates of the Epo receptor were blotted with anti-pY antibodies. (C) Altered downstream Epo receptor signaling dynamics during Epo induction in J2-Lyn+/up cells. Immunoblot analysis of total cell lysates of J2-WT and J2-Lyn+/up cell lines 0, 10, and 30 minutes after Epo stimulation for the signaling molecules indicated and GAB2 immunoprecipitates probed for total phosphotyrosine (pY) and GAB2. Immunoblot analysis was performed on 2 independent experiments producing equivalent results. (D) Increased levels of Epo receptor downstream targets in J2-Lyn+/up cells. Immunoblot analysis of total cell lysates of J2-WT and J2-Lyn+/up cell lines 0, 10, and 30 minutes after Epo stimulation for BclXL, SOCS1, SOCS3, and CIS.
