Anti zfx antibody

CHIP assay was performed using the EZ-CHIP kit (Millipore, Billerica, MA, USA). Briefly, approximately 1 × 10⁷ cells were fixed with 1% formaldehyde and sonicated on ice with 15 s bursts that were repeated 15 times with 15 s intervals. Optimization experiments were performed in order to obtain an optimal length of chromatin fragments with 200–1000 bp of genomic DNA (Figure S3). Target chromatin fragments were enriched with 2 µg anti-ZFX antibody (Cell Signaling, Beverly, MA, USA) and immunoprecipitated with protein G agarose beads. In a parallel experiment, non-immunized rabbit IgG was used as a negative control. In ZFX overexpression experiments, 2 µg anti-FLAG M2 antibody (Sigma, Saint Louis, MO, USA) was applied as a verification approach. After extensive washing of beads, DNA was freed following protease K digestion. DNA was purified and analyzed using PCR with primers encompassing the putative ZFX-binding sites of SET promoter (Table 1). To assess ZFX binding to SET promoter sequences in the luciferase reporter plasmid, PCR was performed using a forward primer targeting the SET promoter sequences and a reverse primer targeting the vector sequences. Following gel electrophoresis, DNA bands were quantified with densitometry analysis.