et al. 3108 mcb.asm.org Molecular and Cellular Biology on May 3, 2019 by guest http://mcb.asm.org/ Downloaded from mcb.asm.org 3113 on May 3, 2019 by guest http://mcb.asm.org/ Downloaded from FIG 4 ORCA associates with Orc2 directly and utilizes WD to associate with ORC, Cdt1, and geminin. (Aa to Af) Binary infections using ORCA baculovirus in combination with Orc1 (Aa), Orc2 (Ab), Orc3 (Ac), Orc4 (Ad), Orc5 (Ae), and Orc6 (Af) in insect cells, followed by immunoprecipitation using ORCA

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... from whole-cell extracts. Note the prominent interaction between Orc2 and ORCA. (B) Direct interaction between ORCA and Orc2. His-ORCA and His-Orc2 were purified on Talon columns, and the two proteins were incubated; then, immunoprecipitation was carried out using Orc2 antibody. (C and D) Reconstitution of the ORCA-ORC complex. Coinfection by baculovirus carrying ORCA plus Orc1, Orc2, Orc3, Orc4, or Orc5 in insect cells and immunoprecipitation of whole-cell extracts with Orc2 (C) or ORCA (D) antibody. (E and F) Binary infection using ORCA baculovirus in combination with Cdt1 and geminin in insect cells, followed by immunoprecipitation using ORCA antibody from nuclear extracts. (G) Schematic representation of various truncation mutants containing a T7 epitope tag at the N terminus of ORCA. (H) IP in U2OS cells expressing various T7-ORCA mutants and Flag-Cdt1 using T7 antibody and analysis of Cdt1 by immunoblotting. (I) IP in U2OS cells expressing various T7-ORCA mutants and YFP-geminin using T7 antibody and analysis of geminin by GFP immunoblotting. Note that only ORCA constructs possessing the WD40 domain efficiently interact with Orc2 (47), Cdt1, and geminin (T7-ORCA.128-647, T7-ORCA.270-647, and T7-ORCA.1-647). The asterisks indicate cross-reacting bands. Shen et al. 3114 mcb.asm.org Molecular and Cellular Biology on May 3, 2019 by guest