FIGURE 2.

Mcb1 localization and its association to MCM. A, cellular fractions were prepared from mcb1HA mcm2V5 (FY4122) according to Fig. S6A. An equal volume (10 μl) of each fraction was separated by 8% or 15% SDS-PAGE and immunoblotted for Mcb1HA, Mcm2V5, Mcm7, α-tubulin (marker for cytosol and nucleoplasm), and Fib1 (Nop1; marker for chromatin). B, Mcb1 interacts with all other MCM proteins except Mcm2. Lysate was prepared from asynchronous mcb1HA mcm2V5 (FY4122) cells with B88 buffer. Twenty micrograms of soluble protein were loaded as input (lane 1). Identical amounts of lysate were precleared and immunoprecipitated (IP) with the antibodies shown. Ten microliters of immunoprecipitated sample (1:7, v/v) were used for each immunoblot. Samples were separated by 8% SDS-PAGE. Soluble lysate was immunoprecipitated with no antibody, anti-HA, anti-V5, anti-Mcm4, and anti-Mcm7 and immunoblotted for Mcm2V5, Mcb1HA, Mcm4, Mcm6, Mcm7, Mcm3, or Mcm5.