Chromatin immunoprecipitation

Data on HSF1 binding to Pmaip1 assessed in genome-wide ChIP-Seq analysis were extracted from the dataset available on Gene Expression Omnibus, accession no. {"type":"entrez-geo","attrs":{"text":"GSE56735","term_id":"56735"}}GSE56735 [34]. The ChIP assay was carried out according to the protocol of a ChIP kit from Upstate Biotechnology (Lake Placid, NY, USA) using protein A-sepharose beads (GE Healthcare Europe GmbH, Freiburg, Germany) or according to the Dynabeads™ Protein A from Life Technologies protocol (Thermo Fisher Scientific, Waltham, MA, USA), or according to the protocol from the iDeal ChIP-Seq Kit for Transcription Factors (Diagenode, Denville, NJ, USA). Standard crosslinking procedure was used. For each IP reaction, 30 µg of chromatin sonicated to 100–500 bp fragments and 3 µg of rabbit anti-HSF1 polyclonal antibody (ADI-SPA-901, Enzo Life Sciences, Farmingdale, NY, USA) were used. For negative controls chromatin samples were processed with IgG from rabbit serum (Merck KGaA) or without antibody. Immunoprecipitated DNA was dissolved into 20 µl of H₂O and analyzed by PCR/qPCR using 1 µl as a template and 40 PCR cycles. In “Input” sample, 0.003–0.006% (ChIP-PCR) or 1% (ChIP-qPCR) of a total material used for the ChIP assay served as a template.