Affinity purification of pRB-associated cellular proteins from HeLa cell nuclear extract. Proteins were bound to a large pocket (amino acids 379–928) GST–pRB construct (GST–RBLP). (a) Verification of pRB-associated APC/C subunits: input, 2% input; pRB, GST–RBLP; GST, GST alone; −/+ NE, HeLa cell nuclear extract. DP1 serves as a positive and Cdc6 as a negative control. (b) pRB-associated proteins were eluted under native conditions and separated by a glycerol gradient. Fractions were taken and analysed for comigrating APC/C subunits. (c) Large pocket GST-fusion proteins of pRB, p107 and p130 were incubated with HeLa cell nuclear extracts to test APC/C binding. DP1 (from HeLa cell nuclear extracts) and E1A (from HEK293 whole-cell extracts) serve as positive controls. (d) Myc-tagged pRB, p107 and p130 (Myc–PP, pocket protein) were transiently cotransfected with untagged Cdc27 in U2OS cells and harvested after 24 h to verify in vivo association. Cdc27 was coimmunoprecipitated using anti-Myc antibodies. Uncropped images of the gels are shown in the Supplementary Information, Fig. S6a–d.