FIGURE 3.

Localization of endogenous aSBDS in a cell lysate. (A) Identification of endogenous aSBDS in different cell fractions by western blot: (1) cell extract; (2) crude ribosomes; (3) high-salt washed ribosomes; and (4) post-ribosomal supernatant (HSW). (B) Density gradient fractionation, after fixation with HCHO, of cell lysates programmed for translation and incubated at 70°C for 45 min. (C) Density-gradient fractionation of: crude 70S (70 pmol) in the 1st panel, HSW 70S (70 pmol) in the 2nd panel, and 50S subunits (50 pmol) in the 3rd panel. Each sample was incubated at 65°C for 20 min in the presence of 1 mM GTP. Braces group experiments made with the same ribosome preparation. The distribution of endogenous aIF6 and aSBDS shown at the bottom of each gradient profile was revealed by western blotting of the individual fractions with the anti-aIF6 and anti-aSBDS antibodies, respectively. In (B,C) the distribution of ribosomal subunits was identified by the optical scans at OD254 nm of the gradients. A representative image of at least three independent sucrose density experiments is shown for each analysis.