Epo induces expression of PDGF-BB in activated endothelial cells, which is partially responsible for Stat5 phosphorylation in SMCs. (A) Effect of PDGF-BB on phosphorylation of Stat5 in SMCs. Serum-starved cells were stimulated for 15 minutes with Epo or PDGF-BB and Stat5 phosphorylation was investigated by Western blotting with phospho-specific antibodies. Total levels of Stat5 and α-tubulin remained unchanged. (B) PDGF-B mRNA expression was induced by Epo in TNFα-activated HUVECs. HUVECS were treated for 4 hours with Epo or TNFα (25 ng/mL) and mRNA expression was investigated with quantitative RT-PCR. (C) Serum-starved SMCs were untreated (lane 1), stimulated with PDGF-BB (lane 2), or treated with the PDGFR-specific tyrosine kinase inhibitor CP-673,451 before stimulation with PDGF-BB (lane 3). SMCs were stimulated for 15 minutes with medium conditioned for 24 hours by HUVECs that were untreated (lanes 4-5) or treated with Epo (lane 6), TNFα (lane 7), or Epo and TNFα (lanes 8-9). SMCs were pretreated with the PDGFR-specific tyrosine kinase inhibitor CP-673,451 before stimulation with conditioned medium from HUVECs that were untreated (lane 5) or treated with Epo and TNFα (lane 9). Phosphorylated and total Stat5 was analyzed by Western blotting. Membranes were reprobed with antibodies against α-tubulin to check equal protein loading.