Verification of putative Elf5 target DNA by independent ChIP and examination of their expression levels in Elf5 null-mouse mammary glands. A. Independent ChIP assay to demonstrate Elf5 occupancy. Mouse mammary gland at pregnancy day 17.5 were immunoprecipitated with anti-Elf5 antibodies and analyzed by PCR using specific primers that amplified genomic DNA segments located close to putative Elf5 target genes and Gapdh as negative control. As positive control of PCR, an aliquot (1%) of chromatin complex before immune isolation was used as input. Nonspecific binding was assessed using goat IgG or no antibody. B. Realtime RT-PCR analysis of Elf5 target genes in WT and Elf5-null mammary gland. Total RNA from wild type (WT) and K14-Cre/Elf5f/f (Elf5 null) mouse mammary glands at pregnancy day 17.5 and lactation day 1 were analyzed for relative expression of mRNA levels of putative Elf5 target genes by real time RT-PCR. Data shown is from at least two independent experiments.