Mutation of the STAT5 binding site Y343 in chimeric EE372 and EE375 receptor forms inhibits EGF-induced SKT6 cell hemoglobinization. (A) To further test roles for STAT5 during ligand-induced SKT6 cell differentiation, the Y343 STAT5 binding site within two distinct truncated chimeric receptor forms, EE372-Y343F and EE375-Y343F, was mutated to phenylalanine. These point-mutated chimeras are illustrated, and levels of transcript expression for these chimeras and endogenous Epo receptors in SKT6-EE372, EE372-Y343F, and -EE375-Y343F cell lines are presented. For EE375-Y343F, increased transcript size (*) is due to expression from the dicistronic vector. (B) To functionally confirm loss of STAT5 signaling via the receptor forms EE372-Y343F and EE375-Y343F, derived SKT6 cell lines were exposed to either EGF (±35 ng/mL) or Epo (±20 U/mL) for 180 minutes, and induced transcription of the STAT5-regulated gene, cis, was assayed by Northern blotting. Equivalence in loading was confirmed by hybridization to a 32P-GAPDH cDNA.
