Protein levels of MeCP2 were determined by western blot analysis. MBH tissue samples were processed for protein extraction followed by quantification of total protein levels by Bradford Assay (Bio-Rad Laboratories, Hercules, CA). About 30 µg of total protein was run in 12% SDS PAGE and transferred to PVDF membrane at 30 V overnight at 4°C. The membranes were blocked in 5% nonfat dry milk-TBS-0.1% Tween 20 (TBST) at room temp for 2 h. The membranes were incubated with primary antibody in the same blocking buffer at 4°C overnight. The primary antibodies used were rabbit anti-MeCP2 (1∶1000; EMD Millipore, Billerica, MA), and mouse anti-actin antibody (1∶5000; Vector labs, Burlingame, CA). The membranes were washed in TBST and then incubated with corresponding peroxidase conjugated secondary antibody at room temperature for 1 h. The membranes were washed in TBST and incubated with ECL reagent and were developed on the film using X-ray developer. The protein band intensities were determined by Image J analysis software (National Institutes of Health, Bethesda, MD). Protein band intensity was normalized with corresponding actin band intensity.
