Localization and function of a eukaryotic-initiation-factor-2-associated 67-kDa glycoprotein

Figure 4 Polysome association of p67 and protein kinase. COS-1 cells were transfected with pETFVA^-/p67 and co-transfected with pETFVA^-/p67 and pETFVA^--/296P. At 48 h post-transfection, the cells were lysed by ribosome binding solution with 0.5% NP-40 and centrifuged at 100 000 g for 1 h. A: The total cell extracts (lanes 1 and 2), supernatants (lanes 3 and 4) and S100 pellets (lanes 5 and 6) were resolved by SDS-PAGE. The levels of ribosome-associated p67 and protein kinase were probed with corresponding antibodies using Western blotting analysis. B: Overexpressed p67 in the total cell extract was affinity absorbed (lane 2) with wheat germ agglutinin (WGA)-agarose. The total cell extract (lane 1) and WGA binding proteins (lane 2) were resolved with SDS-PAGE, and the level of p67 was detected using Western blotting analysis. Deglycosylated p67 was labeled as DG-p67.