Figure S4. Purified proteins used for in vitro assay.

(A) Purified Cdt1-3FLAG, PCNA, and RFC proteins. (B) CRL4^Cdt2 complex after purification on FLAG-resins. The asterisks are contaminants. The purified proteins were Western blotted with antibodies for each protein. (C) The CRL4^Cdt2 complex after purification on FLAG-resins was further purified on glycerol gradient centrifugation. The peak fractions of CRL4^Cdt2 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 CRL4^Cdt2 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.