Fig. 6. Ch-1 fragment can support IRES-mediated, but not uncapped mRNA translation. (A) Schematic representation of RRL incubation and fractionation. (B) RRL (200 µl) was incubated alone, with 15 µl of in vitro translated L protease or with 1.4 µg of HIV-2 PR. After 1 h at 30°C, L and HIV-2 proteases were inhibited by 0.5 mM E-64 or 10 µM Palinavir, respectively, and lysates were ultracentrifuged [see Materials and methods and (A)]. Aliquots of lysate corresponding to 2 µl of parent RRL were resolved on a 10% SDS–PAGE, and western blot analysis was performed with antibodies specific to the C-terminal part of eIF4GI (upper), eIF4E (middle) and p48 protein of eIF3 (bottom). The resulting proteins or fragments observed, and molecular weight markers (in kDa) are indicated on the figure. The asterisk denotes a non-specific reaction with the eIF4GI antibody. CS, supernatant control; CR, ribosomes control; LS, L-treated supernatant; LR, L-treated ribosomes; HS, HIV-2-treated supernatant; HR, HIV-2-treated ribosomes. (C) Different reconstituted lysates were obtained by combining 1 µl of ribosomes and 5 µl of supernatants described in (A). XL–EMCV mRNA (10 ng/µl) was then translated under these conditions. Samples were analysed as described in Figure 3.