Fig. 1.

Differential analysis strategy to identify specific human PIPs. Erythrocytes submitted or not to in vivo cross-linking were lysed, and proteins were purified by overnight incubation at 4 °C with either the MCP21-Sepharose antibody or the OX8-Sepharose antibody as negative control. After extensive washing with 20 mm Tris-HCl, 150 mm NaCl, 1 mm EDTA, 10% glycerol, 5 mm MgCl₂, 2 mm ATP, pH 7.6, proteins were eluted with a saline step using the same buffer containing 3 m NaCl. Two-milliliter fractions were collected and stored at 4 °C. Protein concentration was determined using the Bio-Rad Protein Assay, and proteasome content could be estimated by measuring the in vitro chymotrypsin-like activity as described under “Experimental Procedures.” The fractions containing the most proteasome activity were then further analyzed by Western blotting and by SDS-PAGE followed by trypsin digestion and LC-MS/MS analysis. Proteins identified in each case (experiment and negative control) were subjected to a differential analysis using the MFPaQ software (38) so that a list of specific MCP21-interacting proteins could be generated. ChT-L, ChT-like.