Figure 2. ChIP assay indicates that USF binding to breast apelin promoter in vivo is increased during lactation when compared to cyclic rats. A) Schematic of proximal promoter region of rat apelin gene depicting location of a putative USF binding site and start sites of distal and proximal primers (arrows) used in PCR amplification; –215 and –45 bp = start sites. B) Breast chromatin of cyclic (n=5) and 3-d lactating (n=6) rats was immunoprecipitated with USF-1 antibodies and followed by PCR amplification of promoter region (–215/–45 bp) containing the putative USF binding site. C) Positive input DNA used for PCR was 2% of the amount used for immunoprecipitation. D) Control immunoprecipitation was done with a nonspecific IgG antibody (Ab).