Figure 3.

Coimmunoprecipitation of fission yeast MCMs demonstrates different strengths of association. Identical amounts of FY803 lysate were immunoprecipitated (∼1.5 × 10⁸ cell equivalents; ∼750 μg total protein per immunoprecipitate) with the antibodies shown. Immunoprecipitates were washed nonstringently with lysis buffer (LB) (A, B, C, and D, top panels) or stringently with modified RIPA buffer (RIPA) (A, B, C, and D, bottom panels). Samples were separated by SDS-PAGE. Lane 1: immunoprecipitation with irrelevant antibody (α-tubulin), lane 2: α-Cdc19p, lane 3: α-Mis5p, lane 4: α-HA, lane 5: α-Cdc21p. Identical blots were probed with antibodies to Cdc19p (A), Mis5p (B), HA epitope tag (C), and Cdc21p (D). Open arrows show degradation products of Cdc19p (A) and Cdc21p (D). FY803 is a nda4-HA integrant, and anti-HA antibody was used to immunoprecipitate/immunoblot Nda4p-HA. Similar results were observed for a wild-type strain (our unpublished results).