MCP-1 expression is up-regulated by TGF-β and active ALK5. (A-C) ECs were treated with or without TGF-β1 (5 ng/mL) for 2 hours before being harvested for RNA extraction. To express receptors, ECs were infected with recombinant adenovirus expressing active forms of TGF-β type I receptor ALK1(AAD) or ALK5(AAD) in the presence of 2 μg/mL tetracycline for 16 hours and then incubated in growth medium without tetracycline for 10 hours to turn on receptor expression. Total RNAs were extracted and RT-PCR (A-B) and real-time PCR (C) were performed. MCP-1 expression was quantitated and normalized to GAPDH and relative value expressed in histograms. Three independent experiments have been performed and a representative is shown. Error bars indicate standard deviation (SD). (D) The culture medium of HMVECs after different treatments was immunoprecipitated with anti–MCP-1 antibody. Anti–MCP-1 immunoblotting was carried out. (E) Lysates from different type cells were subjected to anti-ALK5 immunoblotting. Tubulin served as a loading control.