MCM-BP interacts with individual MCM proteins.

<p>A. Affinity tagged MCM4, 5, 6 or 7 (M4, M5, M6, M7) were expressed from baculoviruses in the presence (+BP) or absence of a baculovirus expressing MCM-BP. Cell lysates were then incubated with the appropriate affinity resin and bound protein were eluted by boiling in SDS buffer. Cells infected only with the MCM-BP baculovirus (BP lanes) were included as a negative control for each resin. A Coomassie stained gel of the eluted proteins is shown. B. The indicated MCM protein was expressed in insect cells with or without MCM-BP. Cell lysates were clarified by centrifugation and equal fractions of the soluble (S) and pelleted proteins (P) were analysed by SDS-PAGE and Coomassie staining. C. Affinity tagged MCM4, 6 or 7 were expressed with or without MCM-BP and recovered on affinity resin as in A. The top panel shows the Coomassie stained gel of the eluted proteins. The same samples were also immunoblotted using anti-MCM-BP antiserum (bottom panel). Purified MCM-BP (200 ng) was loaded as a marker in lane 1 (BP-M). D. Strep-tagged MCM2 was expressed in insect cells with or without MCM-BP, recovered on Strep-T actin resin and processed as in C. MCM-BP expressed on its own was also incubated with Strep-T actin resin to establish the level of nonspecific interaction with the resin (lane 1). Purified MCM-BP (1 µg) was loaded as a marker in lane 4 (BP-M). E. The indicated 6-His-tagged MCM protein was expressed with or without MCM-BP (as indicated) and recovered on Ni-NTA resin. In lane 1, MCM-BP was expressed on its own to determine the level of nonspecific binding to the Ni-NTA resin. A Coomassie stained gel of the eluted proteins is shown.