GT1-7 cells or GH3 cells were plated in 15-cm plates, grown to 80–90% confluency and treated as indicated. For animal experiments, hypothalamic tissue was dissected from mice after the indicated manipulations. Cells or hypothalamic tissue were crosslinked with 1% v/v formaldehyde for 10 min at 4°C, and quenched with 0.125M glycine for 5 min. Chromatin was prepared and immunoprecipitations performed, as described previously 49. Immunoprecipitations were conducted using antibodies for CREB (#244, PBL-Salk Institute) and TORC1 (#6937E1, PBL-Salk Institute). Rabbit IgG (Santa Cruz) was also utilized as a negative control. Following crosslink reversal, and proteinase K treatment, DNA was isolated by two rounds of phenol-chloroform extractions and ethanol precipitation. DNA was treated with RNase (DNase-free; Roche) and purified with a QIAquick PCR purification kit (Qiagen). Occupancy on target promoters was determined by PCR or quantitative real-time PCR.
