We performed ChIP assays in isolated chromatin from cultured Neuro2A cells transfected with myc-tagged FoxO1-ADA and whole-brain samples from C57BL/6 mice using the following primers: proximal site, forward, 5′-GGAGTTCAGGATACTC-3′, reverse, 5′-GGATGTCTTCAAGTCCC-3′; middle site, forward, 5′-TTCTCTGAAGGGCTTGGGC-3′, reverse, 5′-CCCACTCACTCCCTGCAT-3′; distal site, forward, 5′-GGGACACAGCTAAAGTGCCC-3′, reverse, 5′-GCAACAGAAGCTAGGTG-3′; non-target site, forward, 5′-GGGTAATCCAGCATGGG-3′, reverse, 5′-AAGCTGGTGGTCCC-3′. The PCR conditions were 95 °C for 3 min and 40 cycles of denaturation at 95 °C for 30 s, annealing at 58 °C for 60 s and extension at 72 °C for 60 s for all primers. We used anti-FoxO1 rabbit polyclonal (Abcam, Cat.No. ab39670 and Chemicon, Cat.No. AB4130) or anti-c-myc mouse monoclonal (Roche, Cat.No. 11667149001) antibodies and protein A/G-coupled agarose beads (Thermo Scientific, CA, USA, Cat.No. 20422) to pull down FoxO1-chromatin complex.
