Cks proteins direct cyclin B1 binding to the phosphorylated APC/C. (A) Cells were treated with siRNA pools targeting Cks1 (siCks1; control, or both Cks1 and Cks2; siCks1 + 2). The cells were thymidine released, blocked in nocodazole, and collected by mitotic shake off. Extracts were blotted with the indicated antibodies. IPs with anti–cyclin B1 antibodies were performed and blotted to examine coprecipitation of the indicated proteins. APC3 binding in cyclin B1 IPs correlates with the hyper-phosphorylated APC3 (APC3-PPP) present in extracts. (B) Cells were treated with siRNA pools targeting Plk1 (siPlk1), both Cks1 and Cks2 (siCks), or Plk1, Cks1, and Cks2 (siPlk1 + siCks). Mitotic cells were collected as in A, and extracts were Western blotted and probed with anti-Cks1 antibody. (top) A background band detected by the Cks antiserum (asterisk) is shown. Serial dilutions of control (ctrl) mitotic cells show that Cks1 knockdown is ∼75% after siCks or between 50 and 75% after siPlk1 + siCks. (C) The same lysates shown in B were used for IP of cyclin B1 and blotted to examine APC/C and Cdc20 binding. Serial dilutions of control cyclin B1 IPs are shown as a reference. Markers are given in kilodaltons. WCE, whole cell extract.