Fig. 1. Analysis of cdc2- and cdc13-YFP proteins. (A) Co-immunoprecipitation assay of either cdc13-YFP or cdc1381-YFP and cdc2p. Cells expressing cdc13-YFP (AD178, lanes 1,3) or cdc1381-YFP (AD203, lanes 2,4) were grown for 20 hours at 32°C in EMM2 without thiamine. Immunoprecipitation from cell extract proteins was performed with polyclonal anti-cdc2p serum. Cdc13p (tagged or untagged) was detected in total cell extracts (lanes 1,2) or immunoprecipitated complexes (lanes 3,4) by western blot using anti-cdc13p mAb. Lanes 1,2, 50 µg of cell extract proteins. Lanes 3,4, immunoprecipitation. (B) pREP5 plasmids with the cdc2- (lanes 1-3) and cdc13-YFP (lanes 5-6) fusions were integrated at the gene locus as shown on the cartoon and strains were grown for 20 hours in either EMM2 without thiamine (-T) or YES (+T) medium. 50 µg of cell extract proteins were loaded and detected using mAbs against cdc2p (1-3) and cdc13p (4-6). Lane 1, pREP5::cdc2-YFPint (AD143) -T; lane 2, pREP5::cdc2-YFPint (AD143) +T; lane 3, pREP5::cdc2-YFPint nmtcdc2::kan^R (AD185) -T; lane 4, control WT strain (PN745); lane 5, pREP5::cdc13-YFPint (AD112) -T; and lane 6, pREP5::cdc13-YFPint (AD112) +T.