miR-130a, but not the other microRNAs with consensus binding sites in the 3′-UTR of the GAX cDNA, is expressed in HUVECs. (A) GAX promoter activity is not decreased by serum or proangiogenic factors. HUVECs were cotransfected with pGAX-Luciferase and pRL-SV (Renilla luciferase, Dual Luciferase Assay System) as described in “Transfections.” Luciferase activity was normalized to Renilla luciferase activity to control for transfection efficiency. Error bars represent SD. (B,C) Expression of 11 microRNAs in HUVECs in low serum and 2% serum. miR-130a is the only microRNA for which expression is detectable in HUVECs, and its expression is up-regulated by exposure to serum in a dose-dependent fashion. Northern blots were performed as described in “Northern blots” to detect these microRNAs. (D) Location of the 2 miR-130a consensus binding sequences in the GAX 3′-UTR.