Salmon Sperm DNA/Protein A agarose 50% slurry. Primary MeCP2 antibody (1∶1000) (EMD Millipore, Billerica, MA) was added to the remaining precleared supernatant and incubated overnight. Equal concentration of the normal Rabbit IgG (Cell Signaling Technology, Danvers, MA,) was employed as a negative control. Rabbit IgG agarose beads were added and incubated by mixing on a rotator at 4°C. Agarose beads were washed, DNA was eluted after reverse cross linking with proteins. Real-time PCR was performed with the resulting sample as well as on the negative control. Serial dilutions of the “input” samples were used as a standard curve. PCR conditions were: 95°C for 2 min, 30 cycles of 95°C 30 s, 60°C for 30 s, 72°C for 30 s, subsequently followed by 72°C for 10 min. The results were normalized relative to the control gene. The control gene sequence is ∼1 kb downstream of the GAPDH transcription start and does not bind MeCP2 in rodents [23]. The primer sequences for the POMC promoter and GAPDH controls were listed in Table 1.
