FIGURE 6.

Immunoprecipitation of wheat germ extracts. Wheat germ extract (1 ml) was immunoprecipitated (IP) with rabbit antisera raised to either recombinant eIF4G or eIFiso4G bound to protein A magnetic beads (Genscript). The precipitated proteins were eluted with 50 μl of 2× SDS-PAGE sample buffer, 20 μl of which was separated on a 10–20% acrylamide gel (Bio-Rad) and transferred to nitrocellulose. Controls of recombinant wheat eIF4E (0.05 μg) and eIFiso4E (0.05 μg) were also separated on the gel. The blots were probed with either mouse monoclonal antibody for eIF4E (8F7) or rabbit polyclonal serum raised to native gel-purified eIFiso4E using a mouse or rabbit 1-h complete IP Western kit (Genscript).