The eIF4E overexpression increases RFLAT-1 protein levels. (a and b) PHA-activated PBLs were lysed at days 0, 1, 2, 3, and 5. Western blot analysis with anti–RFLAT-1 and anti–eIF4E Ab was performed. Relative amount of eIF4E protein was determined by densitometry followed by normalizing to the amount of α-actinin in the lane. Results are representative of three independent experiments (b). (c and d) S2-6 and S2-6-4E cells were transiently transfected with 1 μg of RFLAT-1 expression constructs in the presence of 1 μg pCMV β-gal. eIF4E was induced 48 hours after transfection, and the cells were harvested 24 hours later. Samples were subjected to Western blot analysis with anti–RFLAT-1 and anti-eIF4E Ab. C, no DNA transfection control. The amount of RFLAT-1 protein was determined by densitometry and normalized to Hsc70 in the lane and to the β-galactosidase activity of the sample (d). Results are representative of two independent experiments. *P < 0.05.