Representative EMSA for cooperative binding interaction of
His6-tagged CPBP (1 μg of protein per well) and nuclear protein
(60% ammonium sulfate fraction, 10 μg of protein per well). (A)
5′ end DNA segment (lane 1, free 32P-labeled probe; lane 2,
60% ammonium sulfate fraction; lane 3, His6-tagged CPBP; lane 4,
mixture of lanes 2 and 3). (B) DNA segment with the first TATA
sequence (lane 1, free 32P-labeled probe; lane 2, 60% ammonium
sulfate fraction; lane 3, His6-tagged CPBP; lane 4, mixture of
lanes 2 and 3) and corresponding DNA segment with point mutation in the first
TATA sequence (lane 5, free 32P-labeled probe; lane 6, 60% ammonium
sulfate fraction; lane 7, His6-tagged CPBP; lane 8, mixture of
lanes 2 and 3). (C) DNA with the second and third TATA sequences
(lane 1, free 32P-labeled probe; lane 2, 60% ammonium sulfate
fraction; lane 3, His6-tagged CPBP; lane 4, mixture of lanes 2 and
3; lane 5, mixture of lanes 2 and 3 and postincubation with rabbit polyclonal
anti-His6-CPBP antibody). (D) 3′ end DNA segment
(lane 1, free 32P-labeled probe; lane 2, 60% ammonium sulfate
fraction, 10 μg of protein per well; lane 3, His6-tagged CPBP, 1
μg of protein per well; lane 4, mixture of lanes 2 and 3). Arrows indicate
DNA-binding complex.