Fig. 3. Composition of eIF4F–Mnk1 complexes in Ad late infected cells. 293 cells were transfected with plasmids expressing GST or GST–Mnk1 proteins. At 18 h post-transfection, cells were uninfected or infected with wtAd and harvested at 12, 24 or 36 h post-infection (p.i.). (A) Cells were labeled with [³⁵S]methionine and equal amounts of protein resolved by SDS–15%PAGE and fluorographed. (B) GST fusion proteins were recovered from equal amounts of cell lysates by glutathione–Sepharose chromatography, and immunoblot analysis of associated proteins performed using antisera to eIF4GI, GST (for Mnk1) and eIF4E. (C) Endogenous eIF4GI was immunoprecipitated (eIF4G immunop.) from equal amounts of cell lysates and associated proteins resolved by SDS–10%PAGE. Proteins were detected by immunoblot analysis using specific antisera as above. (D) Equal numbers of uninfected cells and cells infected for 12 h (early Ad infection) or 36 h (late Ad infection) were labeled with 200 µCi/ml of ³²PO₄ in phosphate-free medium for 2 h. eIF4E was recovered from equal amounts of cell extracts by m⁷GTP–Sepharose affinity chromatography, equal fractions resolved by SDS–15%PAGE and autoradiographed (upper panel), or transferred to Immobilon-P (Millipore) and detected by immunoblot analysis with specific eIF4E antisera (bottom panel). Quantitation was performed by digital densitometry.