FIGURE 8.

IBtkα enhances translation by counteracting the Pdcd4 repression of target mRNAs. A, schematic representation of luciferase reporter mRNAs. B, depletion of IBtkα by RNA interference decreases the translation of reporter mRNAs with stem-loop structured or unstructured 5′-UTR. HeLa cells (3 × 10⁶) cells were transfected with IBtk siRNA, Pdcd4 siRNA, or control siRNA or left untransfected (mock). After 24 h, cells were transfected with pCMV-LUC (0.2 μg) or pCMV-SL-LUC (0.2 μg) and serum-starved for 12 h, followed by growth in complete medium (10% FBS) for additional 24 h. The luciferase activity measured in untransfected cells was designated as 100%. Mean values ± S.D. (error bars) of five independent experiments are shown. C, overexpression of IBtkα enhances the translation of reporter mRNAs with stem-loop structured or unstructured 5′-UTR. HeLa cells (3 × 10⁶) were transfected with IBtkα-FLAG, IBtkα-FLAG mutants, or empty vector (4 μg). Subsequent steps were performed as described in B. D, IBtkα RNA interference does not affect the global protein synthesis. HeLa cells (3 × 10⁶) were transfected with IBtk siRNA or control siRNA or left untransfected (mock). The rate of protein synthesis was measured by incorporation of ³⁵S-labeled methionine and cysteine into translated protein and normalized to total protein concentration. E, IBtkα depletion by RNA interference decreases the intracellular amount of Bcl-xL. HeLa cells (3 × 10⁶) were transfected with IBtk siRNA or control siRNA or left untransfected (mock), and 48 h later, cell lysates were analyzed by WB with the indicated antibodies. F, HeLa cells were transfected as described in E, and total RNA was analyzed by real-time PCR to measure the level of the indicated transcripts. Mean values ± S.D. of three independent experiments are shown.