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. 2012 Nov 5;287(53):44212–44220. doi: 10.1074/jbc.M112.401778

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© 2012 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 2. — Tom1 and Dia2 interact with Cdc6. A and B, Tom1 Hect domain and Dia2 interact with Cdc6. Hi5 insect cells were co-infected with GST-Cdc6 and FLAG-Hect or Myc-Dia2 baculoviruses. FLAG-Hect, Myc-Dia2, or GST-Cdc6 protein was immunoprecipitated (IP) with anti-FLAG, -Myc, or -GST antibodies and analyzed by immunoblotting. C, endogenously expressed Tom1 and Dia2 co-precipitate with Cdc6. Total cell lysates from strains expressing FLAG-Tom1 and Myc-Dia2 or FLAG-Tom1, Myc-Dia2, and HA-Cdc6 were immunoprecipitated with anti-HA.11 affinity matrix and immunoblotted with anti-FLAG, -Myc, and -HA antibodies. D, Dia2 binding to Cdc6 is resistant to DNase I treatment. Cell lysate from the strain expressing Myc-Dia2 and HA-Cdc6 strain was treated with 20 units of DNase I (Promega) for 45 min on ice. Cdc6 was immunoprecipitated with anti-HA antibodies and immunoblotted with anti-HA and -Myc antibodies. E, the leucine-rich repeat domain of Dia2 is required for its interaction with Cdc6. Total cell lysates from GST-Cdc6 and Myc-Dia2, FLAG-TPR, or FLAG-ΔN214 baculovirus-infected Hi5 insect cells were immunoprecipitated with anti-GST antibodies and analyzed by immunoblotting with anti-GST, -Myc, or -FLAG antibodies.