Fig. 2. Co-precipitation of Cdc27 and Pcn1 from S.pombe. Protein extracts prepared under non-denaturing conditions from cells transformed with the following expression vectors were incubated with Ni–agarose resin, to pull down Pcn1, and the bound fractions subjected to SDS–PAGE and blotted with anti-HA antibodies to detect the presence of Cdc27^tag. Lane 1, vectors pREP3X, pREP4X; lane 2, pREP3X, pREP4XH6-Pcn1; lane 3, pREP3X-Cdc27^tag, pREP4X; lane 4, pREP3X-Cdc27^tag, pREP4XH6-Pcn1. Note that the cells were grown in minimal medium without thiamine, to derepress the nmt1 promoter in the pREP vectors.