Figure 6. 4E-BP1 overexpression or eIF4E reduction restore rapamycin sensitivity.

(A) REN cells were infected with myc tagged-4E-BP1 WT, LM/AA (unable to bind eIF4E) or with control adenovirus with the indicated Multiple of Infection (m.o.i.). WT and LM/AA HA-4E-BP1 expression was confirmed by WB analysis of the myc tag. Exogenous 4E-BP1 is expressed as the same level as the endogenous protein. Serine 65 phosphorilation of 4E-BP1 is reduced by rapamycin treatment, as expected. (B) Infected cells were treated with rapamycin or left untreated and pulsed with ³⁵S-methionine. Methionine incorporation in newly translated proteins was measured in triplicate. WT 4E-BP1 overexpression restores the rapamycin sensitivity in REN cells. (C) REN cells were transfected with eIF4E siRNA or control. After 48 hrs, total protein were analyzed by WB in order to measure eIF4E downregulation. Densitometric analysis of eIF4E level normalized to β-Actin is reported. (D) REN cells were transfected with eIF4E siRNA or control. Cells were treated with rapamycin and the rate of translation was measured. Rapamycin impairs translation where eIF4E level is reduced.