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. 2015 Feb 6;290(12):8002–8010. doi: 10.1074/jbc.M114.615310

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© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 3. — Ser-1126 phosphorylation and binding of Cdk1-cyclin B1 have opposing effects on separase solubility. A, mutational inactivation of its cyclin B1-binding site decreases the solubility of separase (Sep.). Transgenic HEK293 cells transfected with securin (siSECURIN) or GL2 (siGL2) siRNA and expressing WT separase or ΔCLD were arrested in prometaphase before the solubility of separase was determined by pelleting assay and quantified as described for Fig. 2A. I, input; S, supernatant; P, pellet; αTub., α-tubulin; Sec., securin; H3 pS10, Ser-10-phosphorylated histone H3; AU, arbitrary units; Pel., pelleted separase; Sup., soluble separase. B, precipitation of separase in mitosis is alleviated by the S1126A mutation but aggravated by CLD deletion. Lysates of transgenic, prometaphase-arrested HEK293 cells expressing the indicated separase variants were immunodepleted of securin before the solubility of securin-free separase was determined by quantitative pelleting assay as described for Fig. 2A. IP, immunoprecipitation.