Pcu3p in vitro ubiquitylation activity. (a) Each of the 14 S. pombe UBCs were co-overexpressed as 6 × His-Myc-tagged versions with HA-tagged Pip1p from plasmids. UBCs were bound to Ni-agarose beads and co-purifiyied Pip1p was detected by HA antibodies. (b) Lysates prepared from pcu3-13myc and pcu3-13myc Δcsn1 cells were immunoprecipitated with affinity-purified antisera against Pip1p and precipitates were analyzed for coprecipitation of Pcu3p by immunoblotting with Myc antibodies. (c) Pcu3p-associated ubiquitin ligase activity. Pcu3p complexes were immunoprecipitated from the indicated strains with Myc antibodies and polyubiquitylation assays were performed as described in materials and methods using the UBCs indicated ("3" denotes human UBC3/CDC34). Total lysates used for immunoprecipitation and the resulting Pcu3p immunocomplexes are shown in the lower panels. Pcu3p-Myc is not clearly revolved into the neddylated and unneddylated forms in these gels. (d) Ubiquitination assay with Pcu3p-Myc complexes isolated from the indicated strains were performed with wild-type ubiquitin or a mutant lacking all lysines (Ub-R).