FIGURE 9:

VEGF mRNA is more efficiently destabilized by the phosphomimetic TIS11b S334D mutant. (A) COS7 cells were transfected in 12-well plates with pTarget empty plasmid (Vector) or plasmids encoding WT or mutant TIS11b. AT 48 h after transfection, the transcription inhibitor DRB (10 μg/ml) was added, and total RNA was extracted at the indicated time points and analyzed by Northern blot. The membrane was hybridized to a radiolabeled VEGF 3′ UTR probe and rehybridized to 18S RNA probe for loading control. (B) VEGF mRNA levels were normalized to 18s RNA levels and plotted as a percentage of the initial value against time using nonlinear regression to a first-order exponential decay model. Shown are the mRNA decay rates from three pooled independent experiments. (C) VEGF mRNA half-lives were calculated and compared with the half-life of the transcript in the presence of WT TIS11b. p Values and 95% CIs were determined using an F-test.