Depletion of eIF3-p44 using immobilized GST/4.1R-80 fusion protein.

(A) Purified, bacterially produced GST (lane 2) and GST/4.1R-80 fusion protein (lane 3) were visualized by Coomassie blue staining. (B) Rabbit reticulocyte lysates were incubated without GST (lane 1), with GST (lane 2), or with GST/4.1R-80 fusion protein (lane 3) coupled to glutathione–agarose beads as described in “Materials and methods.” After centrifugation, the supernatants were subjected to SDS-PAGE, and blots were probed with anti-eIF3-p44 or anti-β-actin antibodies. (C) Lysates without treatment (lane 1) or pretreated with affinity-purified GST (lane 2) or GST/4.1R-80 (lane 3) beads were tested for in vitro translation activity as described in Figure 6B.