FIG. 7.
Effect of eIF3 and PABP on Paip1-dependent translation stimulation in vivo. (A) HeLa cells were transfected with the indicated siRNAs, and siRNA 4E-T inverted (15) was used as a control (Ctrl). At 24 h after siRNA transfection, cells were transfected as described in the legend of Fig. 6B using the pTet-HA-Paip1 p45 construct. Cells were placed in medium containing 0 or 300 ng/ml of Tet. Data are expressed as the percentage of the relative increase in induction in cells transfected with control siRNA (set at 100%). Error bars denote the standard error of the mean of three independent experiments. (B) Renilla luciferase mRNA was measured by quantitative RT-PCR from total RNA extracted from duplicate HeLa cells from panel A. Total RNA was DNase treated and reverse transcribed. By using RT reactions, Renilla mRNA was quantified and normalized against β-actin mRNA. The luciferase mRNA ratio is calculated by the ΔΔCT method using the repressed expression (+ Tet) for each siRNA as a reference. Error bars denote the standard error of the mean for three independent experiments. (C and D) Extracts from panel A were subjected to SDS-PAGE and Western blotting (WB) with the indicated antibodies.