FIGURE 6.
Analyses of protein assembly with immunoprecipitation. A, EB90mbp323 (2.8 pmol), Orc1–5 subcomplex (0.2 pmol), His-Orc6 (0.8 pmol), and GST-Cdc6 (1.2 pmol) were mixed on ice, and 1 pmol of biotinylated DS fragment was added in Buffer GD containing 5 mm MgCl2, 40 mm potassium glutamate, 0.01% Triton X-100, 60 ng/μl ovalbumin, and an ATP-regenerating system. They were adsorbed to Dynabeads protein G that was preincubated with anti-EBNA1 antibody. The beads were washed three times with Buffer GD containing 0.1 m NaCl, 0.5 mm ATP, and 0.05% Triton X-100 and then transferred to fresh tubes, followed by boiling in SDS sample buffer. The eluted proteins were detected by Western blotting. The eluted DS fragment (biotinylated) was dot-blotted on a nylon membrane and detected by peroxidase-conjugated streptavidin. B, the amounts of the immunoprecipitated proteins and DS were quantified and expressed in pmol in the bar graph as described in the legend to Fig. 4. C, the levels of the immunoprecipitated molecules indicated were quantified, and the relative level of each protein compared with that of EBNA1 dimer (Ratio) was calculated.