Interaction of GST-eIF4G1 proteins with eIF4E and eIF4A. Approximately 40 pmole of the eIF4G1 proteins (coexpressed with eIF4E in E. coli and purified on glutathione-Sepharose) were diluted to 1.5 mL in TBS/NaCl/Triton and bound on 20 μL of glutathione-Sepharose for 2 h at 4°C. The resin was washed twice with TBS and once with buffer A. The resin was resuspended in 100 μL of buffer B, incubated with 0.2 μg of purified eIF4A (expressed in E. coli) for 1 h at 4°C, washed four times with buffer B, and transferred into fresh Eppendorf tubes. Proteins bound were eluted by boiling with SDS sample buffer, fractionated on SDS polyacrylamide gels, and analyzed by Western blotting, using antibodies against eIF4E, eIF4G, and eIF4A as described (Berset et al. 1998). (Upper panel) anti-eIF4G antibody, (middle panel) anti-eIF4A antibody, (lower panel) anti-eIF4E antibody. (Lane 1) eIF4G11–952, (lane 2) GST, (lane 3) GST-eIF4G1 Δ1Δ2Δ3, (lane 4) GST-eIF4G1 Δ1m2Δ3.
