FIG 1.

Ectopic overexpression of miR-195 inhibits IGF2R translation. (A) Levels of miR-195 (a) and U6 RNA (b) 48 h after transfection of cells with pre-miR-195 as measured by Q-PCR analysis. Values represent means ± standard errors of the means (SEM) of results from three separate experiments. *, P < 0.05 (compared with cells transfected with control scramble oligomer). (B) Representative immunoblots of IGF2R, IGF1R, IGF-binding protein 5 (IGFBP5), glucagon-like peptide 2 receptor (GLP2R), GLP1R, and EGFR as examined by Western blotting of the cells described in the panel A legend. Equal loading was monitored by assessing GAPDH levels. (C) Levels of Igf2r and Igf1r mRNAs in the cells described in the panel A legend. (D) Newly synthesized IGF2R protein in cells overexpressing miR-195. After cells were exposed to l-azidohomoalaine (AHA), cell lysates were incubated with the reaction buffer containing biotin/alkyne reagent; the biotin-alkyne-azide-modified protein complex was pulled down by the use of paramagnetic streptavidin-conjugated dynabeads. (E) Distributions of Igf2r (a) and Gapdh (b) mRNAs in each gradient fraction prepared from the polysomal profile in cells after miR-195 overexpression. Nuclei were pelleted, and the resulting supernatants were fractionated through a 10% to 50% linear sucrose gradient. RNA was isolated from different fractions (fractions 1 to 3, free or/and preinitiation RNA; fractions 4 and 5, monosomes; fractions 6 to 12, polysomes), and the levels of Igf2r and Gapdh mRNAs were measured and plotted as a percentage of each of the total levels of Igf2r and Gapdh mRNA in each sample.