Cdc19p-containing MCM complexes do not change
during the cell cycle. Each strain was shifted to 36°C for 4 h
before harvest and lysis. Lanes 1: cdc10-V50 (G1 phase
arrest, FY562); lanes 2: cdc22-M45 (early S phase,
FY583); lanes 3: cdc17-K42 (late S phase, FY322); lanes
4: cdc25–22 (G2/M phase, FY584); lanes 5: wild-type
asynchronous culture (FY254); lane 6: cdc19-P1 (S phase,
FY243); lane 7: cdc21-M68 (S phase, FY786). (A) Equal
amounts of cell lysate (10 μg total protein) were
immunoblotted with antibodies to fission yeast MCMs (7%
SDS-polyacrylamide gels) or α-tubulin (12% SDS-polyacrylamide gel).
(B) Identical amounts of lysate (250 μg total protein) were
immunoprecipitated with anti-Cdc19p antibody. Immunoprecipitates were
washed with lysis buffer and immunoblotted for fission
yeast MCM proteins, as indicated. (C) Flow cytometry measuring DNA
content (X-axis) versus cell number (Y-axis) was used to verify cell
cycle arrest.