FIG. 5.

Induction of PABP mRNA translation by MCMV is preserved in p70 S6K-deficient cells. (A) Growth-arrested WT or p70 S6K1/K2 doubly deficient primary MEFs (DKO) were mock infected (−), infected (+) with MCMV (MOI = 5), or serum stimulated. At 24 hpi, cultures were metabolically labeled with [³⁵S]Met-Cys for 2 h. Cell-free lysates (top) and PABP immunoprecipitates (bottom) were fractionated by SDS-PAGE and analyzed by autoradiography. The mobility of molecular weight standards (in kilodaltons) is shown on the left. (B) Total protein isolated from MEFs infected as described for panel A was fractionated by SDS-PAGE and analyzed by immunoblotting with the indicated antibodies. S6-P, phospho-specific S6. (C) At 22 hpi, MEFs (infected as described for panel A) were pulse-labeled with [³⁵S]Met-Cys for 2 h with or without rapamycin. Cell-free lysates (top) and PABP immunoprecipitates (bottom) were analyzed as described for panel A. FBS, fetal bovine serum. (D) Samples described in panel C were analyzed by immunoblotting with the indicated antisera. The hyper- and hypophosphorylated forms of 4E-BP1 are indicated.