(a) pRB was induced by tetracycline in a stably transfected SAOS2 cell line (SAOS-tetRB) and protein samples for immunoblotting were harvested at various times after pRB induction. GAPDH, loading control. Uncropped images of the gels are shown in the Supplementary Information, Fig. S6e. (b) pRB-mediated G1 arrest of corresponding samples was monitored by FACS counting BrdU-incorporating S-phase cells after tetracycline treatment of parental SAOS2 cells that do not carry inducible pRB (parental, grey bars), and of pRB-inducible SAOS2 as in a (tet–pRB). (c) pRB–APCcdh1 association was monitored over time by immunoprecipitation using anti-pRB antibodies. The pRB antibody concentration was limiting with regard to extract to obtain equal quantities of immunoprecipitated pRB at the different times. Ig: heavy chain. Uncropped images of the gels are shown in the Supplementary Information, Fig. S6f. (d, e) As controls for the specificity of APCcdh1- and pRB-mediated Skp2 protein turnover, SAOS-tetRB cells were transfected with Flag-tagged Skp2 and treated with either the proteasome inhibitor MG132 or mock treated (d), and with Flag-tagged Skp2 and cdh1 siRNA (e). p44/42, loading control. (f) The rate of Skp2 ubiquitination in vivo was measured in the presence or absence of pRB by transfecting HA-tagged ubiquitin and Flag-tagged Skp2 into SAOS-tetRB cells. Cdh1 siRNA cotransfection served as a control. Immunoprecipitations from MG132-treated cell extracts were carried out using Flag-antibody. Ubiquitin chains were visualized using anti-HA antibodies for immunoblotting (IB).