A kinase‐dependent role for Haspin in antagonizing Wapl and protecting mitotic centromere cohesion A, BGST‐Wapl‐N (A), or transiently expressed Myc‐Wapl.

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A kinase‐dependent role for Haspin in antagonizing Wapl and protecting mitotic centromere cohesion A, BGST‐Wapl‐N (A), or transiently expressed Myc‐Wapl immunoprecipitated from asynchronous cells (B), was subjected to in vitro kinase reaction with 6xHis‐Haspin‐KD, followed by immunoblotting with antibodies to 6xHis, Myc, Wapl‐pT8, or GST. C, DWapl‐N‐GST was subjected to in vitro kinase reaction with MBP‐Haspin‐KD (C), or transiently expressed SFB‐Haspin immunoprecipitated from mitotic cells (D), followed by immunoblotting with antibodies to Flag, MBP, Wapl‐pT8, or GST. E, FITC curves of the binding of MBP‐Pds5B‐N to WT or phospho‐Wapl‐N peptide. Kd is shown (n = 3). G, HMBP‐Pds5B‐N was subjected to pulldown by biotinylated Wapl‐N (WT or pT8) peptide immobilized on Neutravidin resin, followed by immunoblotting with MBP antibodies or streptavidin‐HRP (G). The relative pulldown efficiency was quantified (H; n = 3, unpaired t‐test). IWapl‐N‐GST was subjected to in vitro kinase reaction with 6xHis‐Haspin‐KD. After stringent salt wash, bead‐immobilized GST proteins were used to pull down MBP‐Pds5B‐N, followed by anti‐MBP immunoblotting or CBB staining. JMBP‐Pds5B‐N was subjected to pulldown by GST or Wapl‐N‐GST (WT or T8E) followed by anti‐MBP immunoblotting or CBB staining. K, LHeLa cells were transfected with the indicated siRNAs and plasmids, then were lysed for immunoblotting (K), or treated with nocodazole for 3 h to prepare chromosome spreads for immunostaining (see Fig EV4I). The percentages of mitotic cells with sister chromatids unresolved were quantified (L; n = 3, unpaired t‐test). Data information: Means and SDs are shown (E, H and L). See also Fig EV4. IF THIS IMAGE HAS BEEN PROVIDED BY OR IS OWNED BY A THIRD PARTY, AS INDICATED IN THE CAPTION LINE, THEN FURTHER PERMISSION MAY BE NEEDED BEFORE ANY FURTHER USE. PLEASE CONTACT WILEY'S PERMISSIONS DEPARTMENT ON PERMISSIONS@WILEY.COM OR USE THE RIGHTSLINK SERVICE BY CLICKING ON THE 'REQUEST PERMISSIONS' LINK ACCOMPANYING THIS ARTICLE. WILEY OR AUTHOR OWNED IMAGES MAY BE USED FOR NON-COMMERCIAL PURPOSES, SUBJECT TO PROPER CITATION OF THE ARTICLE, AUTHOR, AND PUBLISHER. EMBO Rep, Volume: 19, Issue: 1, Pages: 43-56, First published: 14 November 2017, DOI: (10.15252/embr.201744737)