FIG. 5.
Human geminin blocks replication of a plasmid mediated by the MRE. (A) The plasmid containing the minimal replicator element was cotransfected with the LANA expression vector as well as human geminin and Cdt1 in the indicated lanes. Western blotting using an anti-myc antibody shows expression of LANA in all four transfections. HA-tagged hCdt1 and human geminin were detected by Western blotting using an anti-HA antibody. (B) Hirt-extracted DNAs from all four samples were digested with BamHI (B) (to linearize) and with BamHI plus DpnI (B+D) overnight. Digested DNA was resolved and transferred to a GeneScreen membrane, followed by hybridization with a 32P-labeled puromycin gene probe. DpnI-resistant bands were quantified using ImageQuant software, and relative densities (RD) of DpnI-resistant bands are shown after normalization with the density of the input lane as 10%.