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. 2011 Oct 17;6(10):e26109. doi: 10.1371/journal.pone.0026109

Figure 1. Scheme of identification of insulator components by iChIP.

Figure 1

(A) The 24×cHS4-core plasmid, which possesses 2 copies of the cHS4c×12-LexA cassettes. (B) FCNLD protein consisting of 2× FLAG-tag, a TEV protease cleavage site, the calmodulin-binding peptide, the nuclear localization signal (NLS) of SV40 T-antigen and the DNA-binding domain of LexA. (C) The 24×cHS4-core plasmid was randomly integrated into the genome of a FCNLD-expressing Ba/F3-derived cell line. To isolate the cHS4-core region and associated molecules, the fixed chromatin extracted from the FCNLD/cHS4-core cells was fragmented by sonication and subjected to immunoprecipitation with anti-FLAG Ab. The isolated chromatin complexes were reverse-crosslinked and subjected to SDS-PAGE, silver-staining, followed by mass spectrometry. (D) Interaction of CTCF with the cHS4-core transgene in vivo shown by ChIP assay with anti-CTCF Ab.