Quantifying MeCP2 Protein Recruitment

NIH-3T3 cells were seeded on coverslips in 6-well plates (25,000 cells per well) and transfected with 2 μg plasmid DNA (pEGFPN1-MeCP2 alone or pEGFPN1-MeCP2 and pmCherry-TBL1X5 (link)) using JetPEI (PolyPlus Transfection). After 48 hours, cells were fixed with 4% (w/v) paraformaldehyde, stained with DAPI (Sigma) and then mounted using ProLong Diamond (Life Technologies). Fixed cells were photographed on a confocal microscope (Leica SP5) using LAS AF software (Leica). The number of co-transfected cells with TBL1X-mCherry recruitment to heterochromatic foci was determined for each MeCP2 construct. In total, 113-125 cells per construct were counted (from three independent transfection experiments). This analysis was performed blind. The total proportion of cells with TBL1X-mCherry recruitment by each mutant MeCP2 protein was compared to WT using Fisher’s exact tests.