Figure 3

Site-specific ChIP on cell culture extracts: Individual detection of Sp1 TFBSs within the egfr promoter region. PCR-based analysis of DNA immunoprecipitated by site-specific ChIP targeting each Sp1 TFBSs within the egfr promoter region. Nuclei obtained from formaldehyde-fixed Osteosarcoma HOS cells were lysed, and chromatin was fragmented by specific enzyme digestion. Chromatin was immunoprecipitated either by normal rabbit IgG antibodies as a negative control or polyclonal Sp1 antiserum. Non-immunoprecipitated chromatin was used as total input control. DNAs from either the input chromatin or immunoprecipitated chromatin were subjected to PCR analysis using the Sp1 TFBSs targeting primers or restriction site flanking primers indicated in Figure 2. PCR of input DNA shows equivalent starting material for the assay. As a negative control, primers amplifying a region within the 3'-UTR of the GAPDH gene were used. In the center of the image, the egfr promoter region with all investigated Sp1 TFBSs and enzyme cleavage sites is shown. Dashed arrows point to the target regions of the respective PCR assay. (A) Lanes 1-16 show PCR products of specific assays for bound TF (Sp1) a(Sp1-a), b(Sp1-b), c(Sp1-c) und d(Sp1-d) (c.f. Figure 2 grey bars). White arrows depict the bound Sp1 binding sites. Templates order: PCR positive control (lymphocyte DNA), ChIP input control DNA, DNA immunoprecipitated with anti-IgG (IP negative control), DNA immunoprecipitated with anti-Sp1. (B) Lanes 17-20 show PCR products of the GAPDH control assay. (C) Lanes 21-35 show PCR products of primer assays for enzyme digestion control (c.f. Figure 2 black bars). Templates order: uncleaved HOS DNA from whole cell lysate (uc-HOS: PCR positive control), DNA samples immunoprecipitated with anti-IgG, DNA immunoprecipitated with anti-Sp1 antibody.