FIGURE 5.

GSK-3β is a target of miR-26a. A, sequence alignment of putative miR-26a targeting site in 3′UTR of GSK-3β shows a high level of complementarily and sequence conservation. B, HASMCs were stimulated with a 1-h stretch. Total and phosphorylated (p) GSK-3β protein levels were analyzed by Western blot for the indicated periods. C, HASMCs were transfected with pSilencer or pSilencer-miR-26a expression construct. GSK-3β mRNA levels were analyzed 24 h after transfection by RT-qPCR (left panel), and total GSK-3β protein levels were analyzed 48 h after transfection by Western blot (right panel). D, cells were transfected with pcDNA-GSK-3β construct, and overexpressions of GSK-3β mRNA (after 36 h) and protein (after 48 h) were confirmed by RT-PCR (upper panel) and Western blot (lower panel) methods, respectively. E, cells were transfected with pcDNA-GSK-3β construct, along with indicated amount of pSilencer-miR-26a construct. GSK-3β protein expression was analyzed by Western blot 48 h after transfection. Gel pictures are representative of three separate experiments. Each bar indicates mean ± S.E. (n = 3).