Fig. 2.

PEA3 binds to the Fgf-10 promoter region. RT-PCR showed that immortalised murine lung endothelial cells expressed both Fgf-10 and Pea3 (primers as in Fig. 1 for Fgf-10 and the cDNA and gDNA controls). Pea3 primers were designed flanking an intron such that they amplified only cDNA (A). Western blotting of 60 μg whole-cell lysate from the same cell line showed a clear band at 70 kDa, corresponding to PEA3 (B). ChIP analysis using the same anti-PEA3 antibody showed that PEA3 binds to the Fgf-10 promoter region (C). Immunoprecipitation with an antibody to RNA polymerase II followed by PCR using primers for GAPDH was used as a positive control for the ChIP assay (upper panel), with IgG and no antibody conditions used as negative controls. Presence and integrity of the ChIP input DNA was confirmed by PCR, using genomic DNA purified from ES cells as a positive control for primer efficiency. ChIP analysis using primers specific for Fgf-10 yielded a specific PCR product both with the anti-RNA polymerase II immunoprecipitate and, more strongly, with the anti-PEA3 immunoprecipitate.