Pcu3p interacts with CSN. (a) Lysates prepared from cells containing Myc epitope-modified Pcu3p, Csn3p, Csn4p, and Csn5p, or HA-tagged Csn1p were fractionated by gel filtration. Fractions were probed with Myc and HA antibodies to determine the elution profiles of the respective proteins. Size standards are indicated. Pcu3p-Myc was not resolved into the neddylated and deneddylated forms on this gel. (b) Lysate from csn1-3HA and pcu3-13myc csn1-3HA cells, which carry epitope-tagged versions of Pcu3p and Csn1p at the endogenous genomic locus, were immunoprecipitated with Myc antibodies followed by immunoblot analysis with Myc and HA antibodies. Co-precipitated Pcu3p-13Myc and Csn1p-3HA are indicated. Specificity controls for the immunoprecipitation include an irrelevant antibody (left panel) and a strain lacking Pcu3p-Myc (right panel). (c) The same strains as used in Fig. 3A were induced to express the indicated 6 × histidine-Myc-tagged CSN subunits. CSN subunits were absorbed on Ni-NTA agarose beads, and co-purification of Pcu3p-13Myc was determined by immunoblotting with Myc antibodies. In the control lane, lysate from nontransformed pcu3-13myc cells was used. Note that Csn3p, while inefficiently expressed in the total cell lysate, is equally retained on Ni-NTA beads, probably due to better accessibility of the 6 × histidine tag.