MiR-107 and miR-128 transfections lead to reduced AGO expression. (A) Microarray analysis of the lysates prior to co-IP shows that different miRNA transfections differentially alter the level of AGO mRNAs. Note that miR-107 and miR-128 transfections cause AGO2 mRNAs to decrease. “*,” P < 0.05; “**,” P < 0.01; using Student's t-test, n = 3 replicates each. (B) Western blot analysis of AGO protein expression after H4 cells were transfected with 25 nM of various miRNAs. Total cell proteins were subjected to SDS-PAGE followed by Western blot analysis using anti-AGO antibody, or anti-β-actin antibody. (C) In a separate experiment, three biological replicates of miRNA transfections were performed for each miRNA and Western blot analysis using anti-AGO and anti-actin. AGO and β-actin protein levels of each individual band were quantified blindly on the Western blot and then normalized to levels of β-actin. Note that AGO protein was knocked down by miR-107 and miR-128. Although equal amounts of total protein were loaded in each well, β-actin levels seemed to be reduced in cells following miR-128 transfections.
