SCFPop protein interactions (1A) SCFPop complex formation at endogenous expression levels. Lysate prepared from wild-type fission yeast cells was used for immunoprecipitation with affinity-purified rabbit antisera directed against SCF subunits as indicated. Precipitates were separated by SDS gel electrophoresis and co-precipitating proteins were detected by immunoblotting with the antisera indicated in each panel. Note that Pip1p, Pop1p, and Pop2p antisera precipitated all five SCFPop subunits (lane 1). The control lane (6) contained an immunoprecipitate prepared with antisera depleted of Pip1p antibodies (flowthrough of the affinity column – FT). Various unspecific bands, some resulting from cross-reactivity of the antibody chains, are indicated by stars. Pcu1p is not resolved into the native and Ned8p-modified forms in this gel system (data not shown). (1B) Co-purification of SCFPop1p-Pop2p subunits in a ~500 kDa complex. Wild-type cell lysate was fractionated by gel filtration on a Sephacryl S300 column, and resulting fractions were immunoprecipitated with Pip1p antisera. Precipitates were analyzed by immunoblotting with the indicated SCF antisera. Size standards are indicated. Immunoglobulin heavy chains are indicated by unfilled asterisks. Other unspecific bands or degradation or modified products are indicated by filled asterisks. (1C) SCFPop protein interactions during the cell cycle. Temperature-sensitive cdc25-22 cells were arrested in G2 by incubation at 37°C for 4 h. Cells were released into mitosis and subsequent cell cycle phases by rapidly shifting to 25°C. Samples were taken after the indicated times and prepared for immunoprecipitation with Pip1p (top panel) or Pop2p (bottom panel) antibodies. Precipitates were probed for co-purification of SCF subunits with the indicated antisera. Pcu1p is resolved into two bands, presumably representing native and Ned8p-modified species. The percentage of septate cells, which represent cells in S phase, was determined at each time point and blotted against time as illustrated in the graph.