Figure 6.

Association of spOrc4p with other Orc subunits in vivo. Sp chromatin was extracted with DNase I (1,000 units/ml, lane 1) or with a solution containing the indicated concentrations of NaCl (lanes 6–8). Extracted chromatin fractions were analyzed by immunoblotting with antibodies against either Orc1p (HA), Orc2p, Orc3p, Orc4p (FLAG), Orc5p, or Orc6p. The chromatin fractions that were incubated with DNase I or extracted with 1 M NaCl were immunoprecipitated with anti-HA antibody (Orc1p, lanes 4 and 9) or anti-FLAG antibody (Orc4p, lane 2). As controls for the immunoprecipitation experiments, the HA peptide (1 mg/ml, lane 3) or the FLAG peptide (100 μg/ml, lane 5) was added during the immunoprecipitation step. Proteins adsorbed to beads were first separated by SDS/PAGE, transferred to nitrocellulose paper, and then immunoblotted with antibodies as described above.