Fig. 3. Geminin prevents binding of Xenopus Mcm proteins to hamster chromatin. Anti-HsMcm2 and anti-XMcm3 antibodies recognize both Xenopus (lane 1, Xenopus egg cytosol) and hamster (lane 2, Triton-extracted CHOC 400 G1-phase nuclei) Mcm proteins, which can be distinguished by their slightly different electrophoretic mobility. The anti-XORC2 antibody does not crossreact with hamster ORC2. (lanes 3-6) CHOC 400 metaphase cells were permeabilized with 80 µg/ml digitonin and incubated for 1 hour at 21°C in LSS (lanes 4,5) or HSS (lane 6) Xenopus egg extracts (supplemented with 100 µg/ml aphidicolin). Soluble or loosely bound proteins were removed by Triton extraction as described in Materials and Methods and the washed chromatin was subjected to immunoblotting analysis with antibodies specific for Mcm2, Mcm3 and ORC2. In lane 5, the LSS extract was supplemented with 2 µg/ml of purified Xenopus geminin. Aliquots of Xenopus egg extract (lane 1) or Triton-extracted CHOC 400 G1 nuclei (lane 2) were run in parallel and served as markers for the mobility of the respective proteins of hamster or frog origin.
