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. 2012 Apr 4;287(22):18500–18509. doi: 10.1074/jbc.M111.327528

FIGURE 5.

FIGURE 5.

Artificial tethering of CTIF to the intercistronic region triggers the translation of downstream cistron in an eIF3-dependent manner. HEK293T cells were transiently transfected with 100 nm eIF3b, eIF3c, or eIF3g siRNAs. Two days later, cells were retransfected with 0.1 μg of tethering dicistronic reporter plasmid pcDNA-F/BoxB/R and 2 μg of effector plasmid pλN-EGFP or pλN-EGFP-CTIF. A, schematic representation of tethering dicistronic reporter plasmid pcDNA-F/BoxB/R, which contains firefly luciferase cDNA as the first cistron, 12 repeats of BoxB sequence derived from bacteriophage λ at the intercistronic region, and RLuc cDNA as the second cistron. B, Western blotting showing the specific down-regulation by siRNAs and the relative expressions of λN-EGFP and λN-EGFP-CTIF. The locations of markers for molecular weight (MW) are indicated on the left of the panel. C, RT-PCR of F/BoxB/R mRNAs. The levels of F/BoxB/R mRNA were normalized to the levels of endogenous SMG7 mRNAs. The normalized levels of F/BoxB/R mRNA in the presence of λN-EGFP were set to 100%. D, translational efficiency of F/BoxB/R mRNAs. RLuc activities were normalized to the firefly luciferase activity. The normalized levels of RLuc activity in the presence of λN-EGFP were set to 100%.