FIGURE 1.

p97 is associated with polysomes. Apoptosis in Jurkat cells was induced by agonistic anti-Fas antibody treatment and extracts processed for linear sucrose density gradient analysis (17.5%–50%) as detailed in the Materials and Methods. (A) Schematic of eIF4GI, p97, and their caspase-cleavage products, for further details see Morley et al. (2005). (B) Density gradient profiles from mock-treated cells or cells treated with anti-Fas antibody for the time periods indicated (top of each panel). Each panel shows the 254-nm absorbance trace recorded during fractionation, with the position of various ribosomal complexes indicated below. Each gradient was divided into 12 fractions, which were processed for the Western blots displayed below each trace to indicate the sedimentation behavior of p97, its caspase-cleaved form p86, full-length eIF4GI, and its caspase-cleavage products. Probing was also carried out for RPS6 to locate ribosomal complexes and tubulin to demonstrate absence of nonspecific aggregation. Positions of all identified protein bands are indicated beside the panels; see Materials and Methods for details on the antibodies used. (C) Density gradient profiles from control cells, or cells treated with anti-Fas antibody for 8 h were done in duplicate as in B, and 10 fractions were taken. For each condition, one lysate aliquot was supplemented with 30 mM EDTA prior to formaldehyde cross-linking to disassemble polysomes (right).