FIG. 5.

TNF-α mRNA is associated with HMW polysomes in pVHL-deficient cells. Cytoplasmic lysates from 786-0 cells that either lacked (VHL⁻) or expressed (VHL⁺) pVHL were fractionated through sucrose gradients, and the RNA prepared from each of the 11 fractions was subjected to Northern blot analysis. (A) Representative TNF-α Northern blotting signals; β-actin mRNA signals were included to assess differences in loading and transfer of samples. (B) Quantitation of the relative abundance of TNF-α mRNA in the indicated fractions prepared from sucrose gradients. TNF-α mRNA signals were normalized to β-actin mRNA signals and then shown as the percent abundance of the TNF-α mRNA in a given RNA subset relative to the total cytoplasmic TNF-α mRNA. (C) For mRNA half-life assessments, ActD (2 μg/ml) was added to 786-0 cells and total RNA prepared at the times indicated. mRNA half-lives were calculated after measurement of Northern blotting signals, normalization to 18S rRNA signals, plotting on logarithmic scales, and determination of the time period required to decrease the abundance of the TNF-α transcript to 50% of the initial signal intensity. Data in graphs represent the means ± standard errors of the means from three independent experiments.