Figure 5

Erythropoeitin activates the JAK and MAPK/ERK pathways. A, Four renal cell lines were starved in serum/growth factor-free media containing 0.1% BSA in normoxia or hypoxia. Cells were stimulated with the indicated concentration of rhEPO. Immunoblotting of protein extracts with indicated antibodies (at left of panel) shows JAK2 and MAPK/ERK pathway components in renal cell lines stimulated with rhEPO in the presence of hypoxia. β-actin is used as a loading control. B, Four renal cell lines were starved in serum/growth factor-free media containing 0.1% BSA in normoxia or hypoxia. Cells were subjected to 1 μM of TG10348 (a JAK2 inhibitor) or 1 μM of U0126 (a MEC inhibitor) for 60 mins prior to the addition of 10 units/mL rhEPO. Ten minutes after exposure of rhEPO, cell lysates were collected and subjected to Western blot analysis with the indicated antibodies. β-actin is used as a loading control. C, Cells are treated with the indicated concentrations of rhEPO in media containing 2% FBS for 24 hrs in normoxic or hypoxic condition. Cell lysates are subjected to Western blot analysis. Western blot analysis shows cyclin D1 was induced and p27 ^kip1 and p21 ^cip1 were down-regulated in renal cells stimulated with rhEPO in the presence of hypoxia. β-actin served as loading control.