Individual homooligomeric SCFPop1p and SCFPop2 complexes (7A) Lysate from a strain harboring epitope-tagged endogenous Pop1p in a Δpop2 background or from the reverse strain containing Pop2p-13Myc in a pop1 deletion strain was immunoprecipitated with affinity-purified anti-Pip1p antibodies (lanes 1 and 2). Immunocomplexes were probed for the presence of associated SCF subunits with the indicated antibodies. Strains containing tagged Pop1p and Pop2p in a wild-type background (i.e. in the presence of their respective binding partners) were used as controls (lanes 3 and 4). Wild-type strains did not show any signal with Myc antibodies (lane 5). The control lane (6) contained precipitates prepared in the absence of Pip1p antibodies (bead control). (7B) Lysate from the strains described above were fractionated by gel filtration and fractions were assayed for the elution profile of Pop proteins by immunoblotting with Myc antibodies. The elution profiles for Pcu1p, Psh1p, and Pip1p are shown for reference (bottom three panels). Size standards are indicated. (7C) Ubiquitylation activity of SCFPop1p and SCFPop2p complexes. SCF complexes were immunopurified with Myc antibodies from lysate of the indicated strains. Ubiquitylation assays in the presence of human E1, UBC3, ATP and ubiquitin were performed as described in materials and methods. Note that a substantial amount of the high molecular weight reaction products migrated in the stacking gel. Controls include immunoprecipitates from wild-type lysates (lane 1) and reactions performed in the presence of mutant ubiquitin lacking all lysine residues (lane 6).