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Fig. 2.
Mapping of CABIN1 interaction domain on HIRA. (A) U2OS cells were transiently transfected with the indicated plasmids and immunoprecipitated (IP) with anti-HA. Lane 1, mock; lane 2, wild-type HA-HIRA and wild-type myc-CABIN1; lane 3, wild-type HA-HIRA only; lane 4, wild-type myc-CABIN1 only; lanes 5 to 8, wild-type myc-CABIN1 and indicated HA-HIRA mutants [lane 5, HA-HIRA(del 439-475); lane 6, HA-HIRA(del520-1017); lane 7, HA-HIRA(421-729); lane 8, HA-HIRA(1-600)]. Schematics are color coded as follows: yellow bars, WD40 repeats; red bars, B domain; blue boxes, C domain. Ab, antibody. (B) U2OS cells were transiently transfected with the indicated plasmids and immunoprecipitated with anti-HA. Lane 1, mock; lane 2, wild-type myc-CABIN1 and wild-type HA-HIRA; lane 3, wild-type myc-CABIN1 and HA-HIRA(del737-963); lane 4, wild-type myc-ASF1a and wild-type HA-HIRA; lane 5, wild-type myc-ASF1a and HA-HIRA(del737-963). Schematics are color coded as in panel A. (C) Insect Sf9 cells were infected with baculoviruses encoding the indicated proteins, and complexes were purified by anti-Flag affinity chromatography. Shown is a Coomassie blue (R250) stain of recombinant proteins. Lanes correspond to following proteins: 1, flag-UBN1; 2, Flag-UBN1 and HisHIRA; 3, Flag-UBN1, HisHIRA, and GST-ASF1a; 4, Flag-UBN1, HisHIRA, GST-ASF1a, and myc-CABIN1; 5, Flag-UBN1, HisHIRA, and myc-CABIN1; 6, Flag-UBN1, HIRA(1-405), and myc-CABIN1. (D) Western blot analysis with indicated antibodies of input lysate (IN), column flowthrough (FT), and elution (E) from panel C.
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