Fig. 4. The association of PA28 with the 26S proteasomes modifies the patterns of peptides generated from proteins. (A) IGF-1 was digested by 26S or hybrid complexes, and peptides generated were separated using a C₈ Vydac column as described previously (Cascio et al., 2001), except for the use of an HP 1100 chromatographer (Hewlett-Packard) in the present studies. (B) FITC–casein (10 µM) was degraded as already described (see Figure 3A), and an aliquot was injected onto a C₁₈ Vydac column equilibrated with 10 mM sodium phosphate (pH 6.8) and analyzed by detection of fluorescence. Under these conditions, protein substrate is degraded and peptide products generated at linear rates (Cascio et al., 2001). Peptides were eluted by a gradient of acetonitrile from 0 to 50% in 100 min. Data very similar to those shown in (A) and (B) were obtained in several different analyses.