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. 2008 Aug 4;28(20):6290–6301. doi: 10.1128/MCB.00142-08

FIG. 5.

FIG. 5.

Binding of CSIG to PTEN mRNA and influence of PTEN 5′ UTR on the expression of a chimeric luciferase reporter construct after CSIG silencing. (A) Schematic presentation of the full-length PTEN cDNA and various transcripts derived from the 5′ UTR, CR, and 3′ UTR used in this study. (B) Whole-cell lysates (100 μg) were prepared from HEK 293 cells, and endogenous target transcripts were detected by RT-PCR assay of the corresponding IP materials; PCR products corresponding to PTEN mRNA were visualized on agarose gel. The PCR product of GAPDH served as a negative Ctrl. (C) Results of a pulldown assay using biotinylated fragments to detect bound CSIG by Western blotting. A 10-μg portion of whole-cell lysates (Lys.) and binding of HuR (positive Ctrl) and α-tubulin (negative Ctrl) to PTEN mRNA were included. (D) A polysomal fraction from HEK 293 cell lysates was prepared and subjected to Western blot analysis to evaluate the presence of CSIG. (E) pGL3, pGL3-5′ UTR, pGL3-CR, and pGL3-3′ UTR-2 plasmid (0.1 μg/ml) were transiently transfected into HEK 293 cells along with the pRL-CMV reporter (5 ng/ml) as a Ctrl. Twenty-four hours later, transfected cells were cotransfected either with CSIG siRNA to silence CSIG or with a Ctrl siRNA for 48 h; firefly luciferase activities were determined and normalized against Renilla luciferase activity. Values represent means ± standard errors of the means (SEM) of the results for five independent experiments. (F) Left panel: pGL3-5′ UTR (0.1 μg/ml), pRL-CMV (5 ng/ml) plasmids, and a different dose of pcDNA-5′ UTR [0 μg/ml (−), 2 μg/ml (+), 4 μg/ml (++), or 6 μg/ml (+++)] were transiently cotransfected into HEK 293 cells for 48 h. Right panel: pGL3-5′ UTR (0.1 μg/ml), pRL-CMV (5 ng/ml) plasmids, and pcDNA vectors expressing PTEN 5′ UTR fragments A, B, C, or D (6 μg/ml) were transiently cotransfected into HEK 293 cells for 48 h; firefly luciferase activities then were determined and normalized against Renilla luciferase activity. Values represent means ± SEMs of the results for five independent experiments.