Purified proteins used for in vitro assay.

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Purified proteins used for in vitro assay. Purified proteins used for in vitro assay. (A) Purified Cdt1-3FLAG, PCNA, and RFC proteins. (B) CRL4Cdt2 complex after purification on FLAG-resins. The asterisks are contaminants. The purified proteins were Western blotted with antibodies for each protein. (C) The CRL4Cdt2 complex after purification on FLAG-resins was further purified on glycerol gradient centrifugation. The peak fractions of CRL4Cdt2 tetramer (calculated molecular weight, 313 kD) are indicated by the dotted line. (D) The Cdt2 complexed with CRL4 purified from insect cells were phosphorylated. The purified CRL4Cdt2 from insect cells were treated with λ-phosphatase (λ-PPase) (+) or not (−), and run on SDS–PAGE gel, stained with CBB. The same samples were run together with whole-cell extract (WCE) prepared from Cdt2-FLAG–expressing cells and blotted with anti-FLAG antibody. Akiyo Hayashi et al. LSA 2018;1:e201800238 © 2018 Hayashi et al.