Zitouni S , Méchali F , Papin C , Choquet A , Roche D , Baldin V , Coux O , Bonne-Andrea C .

	Fig 1. xFbw7α protein is expressed and xCyclin E can be degraded in an Fbw7α-dependent manner in MII-arrested eggs.A. Immunoblot analysis with anti-Fbw7 antibodies of protein extracts from MII-arrested oocytes. ivt: xFbw7α, β and γ isoforms translated in vitro. Asterisks denote non-specific immunoreactive bands. B. MII-arrested egg extracts immunoprecipitated with control IgG (C), or anti-Fbw7 antibodies. C. Stage VI oocytes were microinjected with mRNAs coding for hFbw7α or xFbw7α, or with H2O as a control. 12 hr later, they were induced to mature by addition of progesterone. The oocytes were collected at GVBD, 3 hr after GVBD (+3h) and during metaphase II arrest (MII). The equivalent of two oocytes was loaded in each lane. Oocyte extracts were analyzed with antibodies directed against Fbw7, xcyclin E, xCdc6 or tubulin. Cyclin E levels were quantified using ImageJ software and were normalized to tubulin. D. Immunoblot analysis of progesterone-treated oocytes microinjected with hFbw7α or hFbw7α-R465A mRNAs and collected during meiotic maturation.
	Fig 2. xFbw7α is phosphorylated by PKC on S18.A. In vitro translated [35S]-xFbw7α was incubated in MII-egg extracts prepared with phosphatase inhibitors (left panel). [35S]-xFbw7α-wt was incubated 1 hr in MII-egg extracts prepared with phosphatase inhibitors and an equal amount of sample was subsequently incubated with an excess of lambda protein phosphatase (λP) and analyzed by phosphorimaging (right panel). (B) In vitro translated [35S]-xFbw7α-wt, -S18A, -S119A or -129A were incubated 1 hr in MII-egg extracts. C. Sequence alignment of the Fbw7α N-terminal region from Xenopus and human. D. GST-xFbw7α-wt or -S18A bound to magnetic beads were incubated with PKCα, activated (+) or not (-) and then submitted to immunoblot analysis with anti-pS18 antibodies. Total GST-Fbw7 was detected by Ponceau S staining. (E) GST-xFbw7α wt or S18A were incubated in MII-egg extracts plus phosphatase inhibitors, supplemented (+) or not (-) with Gö6976 and with [γ-33P]-ATP for 1 hr at 23°C and analyzed by immunoblotting and by phosphorimaging for quantification of 33P incorporation (lower panel).
	Fig 3. Dephosphorylation of Ser18-xFbw7α correlates with cyclin E ubiquitylation.A. Xenopus MII-arrested egg extracts were treated or not with Gö6976 and subjected to immunoprecipitation with anti-Fbw7 (Fb) or control antibodies (C) and analyzed by immunoblotting to detect xFbw7α and phosphorylated Ser18 (pS18). B. The same extracts were subjected to immunoprecipitation with anti-xCyclin E (E) or control antibodies (C) and analyzed by immunoblotting with anti-xCyclin E and anti-ubiquitin antibodies.
	Fig 4. Endogenous and ectopic Fbw7α are phosphorylated at Ser18 in M-phase.A. FLAG-Fbw7α was transfected or not in HeLa cells, as indicated. Total extracts of asynchronous cells (AS), cells synchronized in G1/S or arrested in pro-metaphase and recovered by shake-off (M) were analyzed by SDS-PAGE and immunoprobed for pS18-Fbw7α and FLAG-Fbw7α. The migration of Cdc27 was used as a marker of M phase (Cdc27P), and anti-tubulin as a loading control. B. HeLa cell extracts (500 μg) from cells arrested in prometaphase were subjected to immunoprecipitation with anti-Fbw7 or control antibodies and analyzed by immunoblotting to detect phosphorylated Ser18 (pS18). Input 10% (i), supernatant after IP (s). C. HeLa cells grown on coverslips were co-transfected with FLAG-Fbw7α and either GFP- PKCα, PKCβ1, PKCε or PKCδ, as indicated. Coverslips were fixed and stained with FLAG antibodies and DAPI. Scale bar, 10 μm. D. HeLa cells were co-transfected with FLAG-Fbw7α and the different GFP-PKC constructs, as indicated. At 24 hr post-tranfection, cells were harvested and the levels of GFP-PKC, pS18-Fbw7α, FLAG-Fbw7α and tubulin were monitored by immunoblotting.
	Fig 5. PKC phosphorylation stabilizes Fbw7α in mitosis.A. HeLa cells were co-transfected with GFP-PKCδ and FLAG-Fbw7α. Cell extracts were probed for GFP-PKCδ, pS18-Fbw7α, FLAG-Fbw7α, cyclin E and tubulin. B. HeLa cells were co-transfected with FLAG-Fbw7α and either GFP or increasing amounts of GFP-PKCδ and analyzed by immunoblotting, as indicated. C. HeLa cells, co-transfected as in B, were treated with nocodazole and probed for cyclin B to monitor progression into M phase. D. HeLa cells were transfected or not (-) with Flag-Fbw7α-wt, -S18A or -S18E, and Fbw7α levels were analyzed by immunoblotting from asynchronous (AS). E. HeLa cells were transfected with FLAG-Fbw7α-S18A or FLAG-Fbw7α-S18E, treated with cycloheximide (CHX) for the time indicated and analyzed by immunoblotting (upper panel). Fbw7 protein levels of two independent experiments were quantified with ImageJ software and normalized to tubulin. The results were plotted as the relative Fbw7 levels compared to those at the time 0 of CHX treatment (lower panel). F. HeLa cells were transfected as in D and treated with nocodazole (+ noco). G. Extracts from HeLa cells, transfected and treated with nocodazole as in F were supplemented with MG132 and phosphatase inhibitors to be subjected to immunoprecipitations with anti-FLAG and control antibodies (IPc) and analyzed by immunoblotting to detect the presence of ubiquitin and pS18-Fbw7α. H. HeLa cells, co-transfected or not (-) with FLAG-Fbw7α-S18E and HA-Fbw7α-S18A, were synchronized in G1/S or arrested in prometaphase and recovered by shake-off. Cell extracts were analyzed by immunoblotting to detect the presence of Fbw7α. Anti-cyclin E and anti-cyclin B were used as markers of G1/S and M-phase respectively.
	Fig 6. Fbw7 dimerization and cyclin E binding is reduced by a negative charge at Ser18.A. FLAG-and HA-tagged versions of Fbw7α either S18A or S18E were co-transfected in HeLa cells and analyzed for their interaction by immunoprecipitation with HA antibodies. Co-precipitated FLAG-tagged Fbw7α was detected by immunoblotting with an anti-FLAG antibody. The membrane was re-probed with HA antibodies. B. Fbw7 or control immunoprecipitates from HeLa cells transfected or not (-) with FLAG-Fbw7α-S18A or –S18E were mixed with in vitro translated [35S]-Fbw7α-18A or -18E (with no tag in N-terminal), as indicated. Complexes were analyzed by phosphorimaging and immunoblotting. C. In vitro translated [35S]-xCyclin E was first incubated with a recombinant Cdc25B phosphatase to activate its associated Cdk2 partner from the reticulocyte lysate and further incubated in MII-egg extracts with phosphatase inhibitors for 30 min. Phosphorylated cyclin E was mixed with FLAG-hFbw7α-18A or-18E immunoprecipitates. Complexes were analyzed by phosphorimaging and immunoblotting. D. In vitro translated [35S]-FLAG-hFbw7α-465A, -18A or -wt were incubated in MII-egg extracts with phosphatase inhibitors for 60 min and mixed with GST-cyclin E bound to magnetic beads. The mutant hFbw7α-465A that cannot bind cyclin E serves as a negative control. Input represents 10% of the total extract (i), total beads (B). Complexes were analyzed by phosphorimaging and immunoblotting.

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