RUNX1 binding and silencing of the chromosomal region chrX:66,763,324-66,763,383. (a) DNA pulldown with oligonucleotides spanning the chromosomal region chrX:66,763,324-66,763,383. Pulldowns using the same methylated or unmethylated oligonucleotides and nuclear extract from GFs were performed in duplicate for each oligonucleotide and were analyzed separately by liquid chromatography/tandem mass spectrometry. Log2 ratios of all identified and quantified proteins in both experiments were plotted against each other. Proteins binding equally well to both oligonucleotides center around log2(ratio) = 0 and are marked in light blue. Proteins binding significantly better to either the methylated or the unmethylated oligonucleotide were determined by intensity-based outlier statistics, requiring a significance of B < 0.001 in both experiments. These proteins are marked with black dots. (b) β-Galactosidase activity in HEK293T cells transfected with the enhancer free, AR CpG5-7, and AR CpG8-11 reporter vectors. All vectors were transfected in unmethylated form and cotransfected with a RUNX1 expression vector (+RUNX1) or the mock vector as control (−RUNX1). Error bars indicate the SD.