(A) Immunoblotting and 2D electrophoresis. First dimension: EMSA; second dimension: SDS-PAGE. Left: control for EMSA with nuclear protein without the probe; right (FTS-1 complex): protein with the FTS-1 probe. The absence of immunostaining on the left indicates that the investigated proteins do not comigrate with the complexes in the absence of DNA. (B) The participation of CBF-A and KAP-1 in the FTS-1 complex was confirmed by EMSA with their antibodies. The complex (*) was supershifted by the CBF-A antibody as indicated (**) and was abolished by anti–KAP-1 antibody; rabbit IgG did not affect the complex. (C) ChIP of 3T3 chromatin with the anti–CBF-A antibody or rabbit preimmune serum (–). Western blotting (upper panel): the immunoblots were probed with antibodies against KAP-1 and CBF-A; the stained IgG is shown as a loading control. PCR (lower panel): Increasing amounts (2, 5, and 10 μl) of DNA eluted from the immunoprecipitates were used as template in PCR with primers encompassing the FTS-1 site in both directions, producing a fragment of approximately 280 bp. Genomic DNA from 3T3 fibroblasts was used in the PCR as a positive control. M, DNA size ladder in kilobases (kb). (D) After induction of EMT, MCT cells showed increased nuclear colocalization of KAP-1 and CBF-A. MCT epithelia were stimulated for 5 days with TGF-β/EGF in culture. Cells were stained with nuclear propidium iodide dye (red) followed by indirect immunofluorescence with primary antibodies against CBF-A or KAP-1 or with control FITC–anti-rabbit IgG alone. Original magnification, ×630.