MiR-24 specifically interfered with activin signaling. RT-PCR (A) and Western blot analysis (B) of hALK4 expression in HEK293 cells transfected with various miRNA vectors as indicated. Quantitation of hALK4 mRNA levels after normalization to GAPDH and hALK4 protein levels to β-actin from 3 independent experiments were shown. (C) HEK293 cells transfected with indicated vectors were treated with activin A (25 ng/mL) for 1 hour and then harvested for anti-phospho-Smad2 Western blot analysis. Smad2 expression was examined with anti-Smad2 immunoblotting to verify protein expression. (D,E) HEK293 cells were cotransfected with ARE-luciferase and FoxH1 (D) or CAGA₁₂-luciferase (E) along with indicated miRNA vectors, and treated with activin A (25 ng/mL) or TGF-β (1.25 ng/mL) for 16 hours before harvested for luciferase assay. (F) HEK293 cells were transfected with CAGA₁₂-luciferase and active ALK4 (caALK4) with indicated miRNA vectors. Luciferase assay was performed at 48 hours after transfection. Reporter assay was performed in triplicate, and the data represent the mean plus or minus SD of 3 independent experiments after normalized to R reniformis activity.