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. 2010 Jan;16(1):239–250. doi: 10.1261/rna.1795410

FIGURE 4.

FIGURE 4.

Changes in PABP expression and activity modulate miRNA repression. (A) miR-30-mediated repression was evaluated in 293T cells transfected with increasing amounts of myc-PABP expression plasmid. The repression level in cells transfected with control vector and miR-30 plasmids was set to 100% and then compared with repression data from cells similarly transfected with miR-30 and PABP (wild-type and M161A) expression plasmids. The total amount of transfected cDNA was kept constant between samples. Western blot analyses were carried out to determine PABP and tubulin levels. Myc-tagged PABP is visible as a distinct band that migrates slightly above endogenous PABP. (B) Northern blot analysis of target mRNA after myc-PABP overexpression. (C) miR-30 activity was evaluated in the context of Paip2 overexpression. Cells were transfected with increasing amounts of Flag-Paip2 plasmid, and repression of miR-30 target mRNA was examined as in A. Flag-Paip2 migrates slightly above endogenous Paip2. (D) miR-30 repression levels during knockdown of Paip2 along with Western blots for Paip2 and tubulin are shown. (E) miR-155 repression of the BACH1 3′ UTR reporter upon PABP, PABP (M161A), and Paip2 overexpression. (F) qPCR of the indicated miRNAs after control or myc-PABP expression plasmid transfection. This experiment was repeated three times, and a representative experiment is shown.