FIGURE 2.
Determination of translational efficiency and de novo protein synthesis. Confluent monolayers of HK-2 cells were growth-arrested and exposed to TGF-β1 (1 ng/ml) for 12 hours before extraction of the cytosol and separation of mRNA on a sucrose gradient as described in Materials and Methods. A: Subsequently, a Northern blot of the fractionated mRNA was probed for TGF-β1. Fractions 1 to 11 represent tRNAs, free ribosomal subunits, monosomes, and untranslated mRNAs in the form of messenger ribonucleoprotein particles. Fractions 12 to 22 represent polysomes of increasing size. B: Graphical representation of the polysome distribution of TGF-β1 mRNA, expressed as percentage of the total TGF-β1 mRNA detected on a given blot in each fraction, with control shown as shaded area and TGF-β1-stimulated as the unshaded area. C: Incorporation of radioactive amino acids into newly synthesized protein by metabolic labeling was used to assess de novo TGF-β1 synthesis. Cells were stimulated with TGF-β1 (0 to 5 ng/ml) in the presence of 40 μCi of 3H-radiolabeled amino acid mixture (1000 μCi/ml; Amersham). Supernatant samples were subsequently collected for TGF-β1 immunoprecipitation, and radiolabeled TGF-β1 was detected by autoradiography.