Figure 9

Effect of in vitro methylation on the ACVRL1 promoter activity. (A, B) Different reporter constructs were subjected to treatment with M.Sss I in the presence or absence of the substrate S-adenosyl-methyonine. Both mock-methylated and methylated constructs were transfected in HEK293T and HMEC-1 cells and the luciferase activity was measured. (***p < 0.005; ns = not significant). (A) Analysis of the different pALK1 reporter constructs. Results are shown as fold changes of luciferase activity (B) Analysis of a TATAbox minimal promoter (see Methods), the Id1 promoter construct and the -1035/+210 pALK1 reporter construct. Activities of untreated samples were given the arbitrary value of 100% and the activity of treated samples is indicated as percentage. (C) Electrophoretic mobility shift assay (EMSA) of the radiolabelled -89/-56 probe containing two Sp1 functional sites. Competition was carried out using the unmethylated (U) or the in vitro methylated (M) probe, as indicated.