Skip to main content
. 2011 Sep 8;286(50):43394–43404. doi: 10.1074/jbc.M111.235127

FIGURE 5.

FIGURE 5.

GSK-3β is a target of miR-26a. A, sequence alignment of putative miR-26a, and its targeting site on the 3′-UTR of GSK-3β shows a high level of complementarity and sequence conservation in vertebrates. B, total RNA and protein were isolated from the ASMCs of Des+/+ and Des−/− mice. GSK-3β mRNA levels were determined by qPCR. Total and phosphorylated (p) GSK-3β protein levels were determined by Western blot. C, ASMCs of Des+/+ mice were transfected with pSilencer or pSilencer-miR-26a expression construct. GSK-3β mRNA levels were determined 36 h after transfection by qPCR, and total GSK-3β protein levels were determined 48 h after transfection by Western blot. D, ASMCs of Des−/− mice were transfected with anti-miR-26a or NS-miR. GSK-3β mRNA levels were determined 36 h after transfection by qPCR, and total GSK-3β protein levels were determined 48 h after transfection by Western blot. E, ASMCs of Des−/− mice were transfected with the GSK-3β 3′-UTR-luciferase construct (luc-GSK-3β-3′UTR) with or without anti-miR-26a or NS-miR. Forty-eight hours after transfection, cells were collected, and then firefly luciferase activities were estimated and normalized to Renilla luciferase activities. F, ASMCs of Des−/− mice were transfected with pcDNA or pcDNA-GSK-3β expression construct. Forty-eight hours after transfection, cell size was determined. G, ASMCs of Des+/+ mice were transfected with pSilencer or pSilencer-miR-26a expression construct along with or without pcDNA-GSK-3β expression construct. Cell size was determined 48 h after transfection. Gel pictures are representative of three separate experiments. Each bar indicates mean ± S.E. (error bars) (n = 3). *, p < 0.05.