FIGURE 8:

The phosphomimetic TIS11b-S334D mutant is more potent than WT TIS11b in decreasing VEGF 3′ UTR–driven luciferase activity and endogenous VEGF mRNA steady-state levels. (A) COS7 cells were cotransfected with pLuc 3′ UTR and pTarget plasmids encoding WT TIS11b, TIS11b-S54A, TIS11b-S54D, TIS11b-S334A, or TIS11b-S334D mutants. Firefly/Renilla luciferase activities of cell lysates were measured as described in Materials and Methods. Results are expressed as relative light units of firefly luciferase activity over relative light units of Renilla luciferase activity and are represented as a percentage of the luciferase activity in control cells transfected with empty pTarget plasmid. Transfections were performed in triplicate, and values are means ± SEM from seven independent experiments. The lower panel is a representative Western blot analysis of overexpressed TIS11b proteins showing that equivalent amounts of overexpressed TIS11b were recovered. (B) Northern blot (NB) analysis of endogenous VEGF mRNA in COS7 cells transfected as in A. (C) Western blot (WB) analysis of TIS11b protein expression levels in the COS7 cells used for the Northern experiment shown in B. (D) Quantification of VEGF mRNA steady-state levels in four independent experiments (means ± SEM). Asterisks: significantly different from WT with *p < 0.05 and **p < 0.01 (one-way ANOVA).