rHuEpo does not activate EpoR signaling pathways in endothelial cells. Endothelial cells (EC) were exposed to vehicle (V), GFC, or rHuEpo (EPO) for 5 (top panel) or 30 minutes (bottom panel). Cell lysates were analyzed by immunoblotting using antibodies specific to phospho-p90RSK (p-90RSK), phospho-AKT (p-AKT), phospho-ERK1/2 (p-ERK1/2), phospho-S6 (p-S6), and eIF4E (loading control). Because of p-S6 being further downstream in the signaling cascade, an extended treatment time of 30 minutes was needed to evaluate phospho-S6. Note lack of signaling in: (A) HUVEC cells, (B) HMVEC-LBI cells, (C) HMVEC-dBI cells, and (D) HCAEC cells when treated with rHuEpo.