Lack of rHuEpo-induced signaling in cardiac, neuronal, and renal cell types. Signaling analyses in (A) rat neonatal cardiac myocytes, (B) primary human RPTECs, and (C) human neuronal cell line SH-SY5Y. Cells were serum deprived overnight and stimulated with 10 U/mL rHuEpo or a volume equivalent of vehicle for up to 30 minutes, with UT-7/Epo cells serving as a control for rHuEpo activity. Positive control cytokines to confirm receptors and pathways were intact for signal transduction included: (A) rMsIFN-γ; (B) rHuIFN-γ in addition to a GFC of rHuEGF, rHuIGF and rHuHGF; and (C) rHuIFN-γ and rHuEGF. Phosphorylation of EpoR downstream signaling proteins ERK1/2, AKT, and STAT5 were evaluated by immunoblot analysis in addition to total amounts of signaling protein and β-tubulin as a loading controls. Experiments were repeated a minimum of 3 times with independent preparations of each cell type with similar results. Note induction of phosphoproteins in response to positive control cytokines and the absence of signaling in response to rHuEpo.