Fig. 7.

Pac2 complexes in vivo. a Western analysis of 11 40–10% glycerol gradient fractions blotted with anti-GFP antibodies to detect ^GFPPac2 (MW 89 KDa); and with anti-α-tubulin (MW 50 KDa); with anti-Rpn10 (MW 30 KDa) and anti-Rpn12 (MW 32 KDa) to detect the 19S RP; and with anti-Pre6 (MW 28.5 KDa) antibodies to detect the 20S CP. Graphs show relative amount of each protein in the different fractions. b Comparison of the Pac2 complexes from Fraction 4 and Fraction 9. ^GFPPac2 was immunoprecipitated with anti-GFP antibodies, and the bead fraction was separated by SDS-PAGE for Western blotting with anti-GFP, anti-α-tubulin, anti-Rpn10, anti-Rpn12, and anti-Pre6 antibodies. c Immunoprecipitation of aliquots of Fraction 4 with anti-Rpn12 (αR12 lane) to immunoprecipitate the 19S RP or anti-GFP antibodies (αGFP lane) to immunoprecipitate ^GFPPac2. Components of the complex that co-immunoprecipitated in each fraction were detected by Western blotting with the antibodies used in Fig. 8a. Controls (C) were Protein A beads without antibody; 10% is a TCA precipitation of an aliquot of the gradient fraction