The rapid down-regulation of GAX expression after exposure to mitogens requires the miR-130a sequences in the GAX 3′-UTR. (A) GAX mRNA expression is down-regulated by mitogens. Quiescent HUVECs incubated in LSM (0.1% FBS) were exposed to mitogens or conditioned medium from MCF7 cells, incubated overnight, and then harvested for isolation of total mRNA, which was subjected to real-time QRT-PCR. Mitogens strongly down-regulated GAX mRNA expression. (B) Mitogens down-regulate endogenous GAX protein expression. HUVECs were incubated in either 0.1% FBS or 10% FBS overnight and then harvested for total protein, which was subjected to Western blot with anti-Flag antibody. Bands were subjected to densitometry and normalized to α-tubulin levels miR-130a. (C,D) Down-regulation of GAX by mitogens depends on the presence of miR-130a sequences in its 3′-UTR. HUVECs were transfected with either pcDNA3.1-GAX-3′-UTR or pcDNA3.1-GAX, incubated overnight in 0.1%, 2%, or 10% FBS, and then fixed for immunofluorescence (C) or harvested for Western blot (D) with anti-Flag antibodies. In panel C, the photomicrograph was taken at 400× magnification; fluorophores and confocal microscope are described in “Immunofluorescence.” Removing the 3′-UTR sequence almost completely abolishes the down-regulation of GAX attributable to serum stimulation. Error bars represent SEM.