Figure 6.
MCMs do not associate with HA-Cdc18p under conditions in which an MCM complex forms. Wild-type fission yeast cells (FY254) were transformed with a plasmid expressing Cdc19p-HA, grown with thiamine (promoter repressed, lanes 1, 3, 5, and 7), or a plasmid expressing HA-Cdc18p, grown without thiamine (promoter induced, lanes 2, 4, 6, and 8). Lysates from each strain were immunoprecipitated with anti-HA antibody. Immunoprecipitates were washed with lysis buffer and immunoblotted with antibodies to Cdc19p (lanes 1 and 2), Mis5p (lanes 3 and 4), Cdc21p (lanes 5 and 6), or HA epitope (lanes 7 and 8), as indicated. Approximately 1.5 × 108 cell equivalents (∼750 μg total protein) of cell lysate were used for each immunoprecipitation.