FIGURE 3.

TRIF increases the translation efficiency of RTA mRNA. A, 293T cells were transfected with cDNA, Flag-RTA (0.1 μg), and TRIF plasmid (0.05, 0.1 μg). The cells were labeled with [S³⁵]methionine for 30 min, and the cells lysates were used for immunoprecipitation with FLAG antibody. The immunoprecipitates were separated and transferred onto Immobilon membranes. S³⁵-labeled protein (newly synthesized) were measured. The membranes were then used for Western blots with RTA-antibody. The relative translation efficiency was calculated as newly-synthesized protein (S³⁵-labeled) versus total proteins. Bottom numbers, the relative translation efficiency of RTA. B, average of relative translation efficiency of RTA from three independent experiments is as shown. Standard deviations are also shown. C, 293T cells were transfected with RTA or TRIF plus RTA plasmids. Cycloheximide (CHX) was added. Cell lysates were made at various time points after treatment (in hours). Western blot was performed to examine the protein stability. The images in the same box indicate that they are derived from the same membranes. D, different RTA-expression plasmids were transfected with various amounts of TRIF plasmids. Total RNA were isolated, and RTA mRNA levels were detected by semi-quantitative RT-PCR. Actin levels were used as a control. One representative of several independent experiments is shown.