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. Author manuscript; available in PMC: 2011 Jan 1.
Published in final edited form as: Nat Cell Biol. 2010 Jun 27;12(7):686–695. doi: 10.1038/ncb2071

Figure 8. JNK-mediated phosphorylation of Cdh1 affects cell cycle progression.

Figure 8

(A) Nuclear (N) and Cytosolic (C) fractions produced from HeLa cells, expressing either control or pEF-FLAG-JNK2α2-ΔKEN plasmids, were synchronized by a DTB and release (0; early S-phase arrest and 6 h; G2 phase) and analyzed by Cdh1 immunoprecipitation followed by Cdh1 and phospho-ThrPro (pTP) immunoblotting. B23/nucleophosmin and PI3K serve as nuclear and cytoplasmic markers, respectively. (B) Immunoblot analysis of Cdc20, Plk-1, and cyclin B1 protein levels in cells expressing for 36 h either Cdh1 wild-type (wt) or non-phosphorylatable triple mutants: (i) Threonine 32, Serines 36 and 151 to Alanine (a protein that cannot be phosphorylated by JNK) or (ii) Serines 40 and 70 and Threonine 121 to Alanine (a protein that is significantly less phosphorylated in vitro by Cdk2, see Figure S21). (C) Flow cytometry cell cycle analysis performed in HeLa cells overexpressing the indicated constructs. The percentage of cells in G1, S, and G2/M (a mixed population of G2 and mitosis) phases of the cell cycle are included. (D) HeLa cells infected, with either control or JNK1/2 shRNAs for 24 h, were cell cycle-synchronized by a double-thymidine block (DTB). Cells were also transfected after the first thymidine treatment with either control or Cdh1 shRNAs. Levels of JNK, Cdh1, and tubulin were assessed at time zero before release (bottom panels) and changes in cyclin B1 and Plk-1 levels and tubulin (as loading control) were analyzed during a cell cycle kinetic of 18 h. (E) Extracts from MEFs isolated from either wild-type or JNK1/2 DKO animals were analyzed by immunoblotting using antibodies directed against major cell cycle regulators (as shown), under downregulation of Cdh1 for 36 h by means of shRNA where indicated. In this figure, JNK2 displays as a 54kDa band (*) while JNK1 displays as a 46kDa band (+).Uncropped images for key results of this figure are shown in Figure S7.