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. Author manuscript; available in PMC: 2016 Nov 16.
Published in final edited form as: Cell Rep. 2016 Sep 20;16(12):3359–3372. doi: 10.1016/j.celrep.2016.08.058

Figure 5. Cyclin F regulates Cdh1 degradation through binding to canonical Cy motif sequences.

Figure 5

(A) Schematic of Cdh1 showing the position of the WD40 repeat domain and 12 putative Cy-motifs. (B) Truncated versions of Cdh1 were tested for binding to Cyclin F by coIP in transfected 293T cells treated with MG132 prior to IP. (C) Myc-Cyclin F and FLAG-Cdh1 (WT and mutants) were expressed in 293T cells, and binding was analyzed by IP following treatment with MG-132. (D) Myc-Cyclin F, Flag-Cdh1WT and FLAG-Cdh1CM1-3 were expressed together in 293T cells and Cdh1 degradation was examined. The non-binding Cdh1CM1-3 mutant is resistant to Cyclin F mediated degradation. (E) FLAG-Cdh1, Myc-Cyclin F and 6HIS-ubiquitin were expressed together in 293T cells. 6HIS-ubiquitin was isolated under denaturing conditions and endogenous Cdh1 was analyzed by immunoblot. Cells were treated with MG132 prior to harvesting. (F) Human Cyclin F was expressed in a yeast strain where endogenous yeast Cdh1 was tagged with Myc.