Chromatin immunoprecipitation assay (ChIP)

ChIP assays were performed using a chromatin immunoprecipitation assay kit following the manufacturer’s instructions (Millipore). Briefly, chromatin was extracted from tissue or cells, sonicated and immunoprecipitated with antibodies against PML, p53 or isotype-matched control IgG. PCR was run on the chromatin and the products were analyzed on a 2% agarose gel. Relative DNA binding was determined by classical qPCR.^47,48 The following primers were used:

Primers for human ChIP

NF1-For: 5’- TAACTTCCAACTCCGGGAGCA-3’

NF1-Rev: 5’- GAGGTGACGTCATCTAACTCCT-3’

GAPDH-For: 5’CGGGATTGTCTGCCCTAATTAT-3’

GAPDH-Rev: 5’GCACGGAAGGTCACGATGT-3’

Primers for mouse ChIP

NF1-For: 5′-AGCCTCAGGACACCCTAACTTCTA-3’

NF1-Rev: 5′-GCGGGTCTCCCCACCTAAACT-3’

Gapdh-For: 5′-ATCCACGACGGACACATTGG-3′

Gapdh-Rev: 5′-TGGTGCTGCCAAGGCTGTGG-3′