Mapping of HIRA interaction domain on CABIN1. (A) U2OS cells were transiently transfected with the indicated plasmids and immunoprecipitated with anti-myc. Lane 1, wild-type myc-CABIN1 and wild-type HA-HIRA; lane 2, wild-type myc-CABIN1; lane 3, wild-type HA-HIRA; lane 4, myc-CABIN1(1-941) and wild-type HA-HIRA; lane 5, myc-CABIN1(942-2220) and wild-type HA-HIRA; lane 6, myc-CABIN1(401-2220) and wild-type HA-HIRA. (B) Insect Sf9 cells were infected with baculoviruses encoding His-HIRA, GST-UBN1(1-175), and Flag-ASF1a, and recombinant complex was purified by nickel affinity chromatography. Shown in the figure is a Coomassie blue (R250) stain of recombinant proteins. (C) Western blot of complex from panel B with indicated antibodies. (D) The trimeric protein complex from panel B was incubated with lysates from U2OS cells transfected with wild-type myc-CABIN1, myc-CABIN1(1-941), or myc-CABIN1(1-400), as indicated. Bound proteins were Western blotted as indicated. (E) Schematic of the HUCA complex based upon binding studies reported here and previously published literature.
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