We carried out chromatin immunoprecipitation assays30 by postfixing cells in 37% formaldehyde and then pelleted them before lysis and sonication. We reserved one tenth of the sample as ‘input’ to quantify the amount of DNA before immunoprecipitation. For the remainder of each sample, extracted chomatin was immunoprecipitated using rabbit polyclonal antibody to NGFI-A (antibody) or normal rabbit IgG (nonspecific; both from Santa Cruz Biotechnology). All of the DNA was then uncrosslinked and subjected to qRT-PCR, using primers directed at the luciferase gene immediately downstream of the transfected NR3C1 promoter (sense, 5′-AGA GAT ACG CCC TGG TTC C-3′; antisense, 5′-CCA ACA CCG GCA TAA AGA A-3′; Tm = 54 °C). The signal for each sample was calculated by dividing the value of the antibody by the input. Each resulting value was multiplied by a constant (1 × 106) to plot the values obtained from the experiments on logarithmic axes.
