Figure 5

Chromatin immunoprecipitation assay of GATA3 in MCF7 breast cancer cells. Chromatin complexes were crosslinked in vivo with formaldehyde (see the Materials and methods section). The GATA3-associated DNA fragments were immunoprecipitated (IP) with mouse monoclonal antibody against GATA3 (HG3-31). DNA samples were isolated before IP (lane labeled 'input'), after elution the unbound proteins from the Protein A–Sepharose batch (lane labeled 'eluted') and after specific IP (lane labeled 'ChIP', for chromatin immunoprecipitation). (a–c) PCR was performed with (a) flanking primers amplifying a GATA1 binding site on the MUC1 promoter (186 bp; see Figure 2), (b) flanking primers for amplification of exonic sequences of the HLA-DQA1 gene (242 bp; negative control), and (c) flanking primers for amplification of the GATA3-binding site on the MUC1 promoter (292 bp) as shown in Figure 2. (d–e) Second ChIP (reChIP) assay using the first eluted DNA–protein complex as input sample. (f) Western blot analysis of GATA3 protein expression: a sample of MCF7 protein extract was run on a 10% SDS-PAGE gel and assayed for GATA3 expression with the monoclonal antibody used in ChIP assays (HG3-31). The expected wild-type band of 48 kDa was immunodetected. Lane WM shows an SDS-7B prestained SDS-molecular mass standard (Sigma-Aldrich).