Repression of α-actinin expression impaired agrin-induced AChR clustering. (A) HEK293 cells were transfected with GFP-α-actinin along with indicated miR-RNAi constructs of α-actinin. Thirty-six hours after transfection, cell were lyzed and resulting lysates were immunoblotted with antibodies against GFP. miR-actinin791 showed the strongest inhibition of α-actinin expression. (B) Young C2C12 myotubes were transfected with the control vector or miR-RNA. After 24 hrs the myotubes were treated with agrin for 18 hr. AChR clusters were examined in myotubes expressing GFP that was encoded by the miR-RNAi parental vector. Agrin-induced AChR clusters were reduced in myotubes expressing miR-actinin791, but not miR-control encoding a scrambled sequence. Bar, 20 μm. (C) Quantitative analysis of data in (B). Data were presented as mean ± SEM, n = 20 each group; **, P < 0.01.