Rabbit polyclonal anti pml

Immunoblots were performed as previously described [50 (link)]. The following primary antibodies were used: rabbit polyclonal anti-SOX9 (1:2000 dilution; Millipore, Burlington, MA, USA), mouse monoclonal anti-STAT3 (1:1000 dilution; Cell Signaling), rabbit monoclonal anti-phospho STAT3 (Tyr705) (1:1000 dilution; Cell Signaling), rabbit polyclonal anti-PML (1:1000 dilution; Bethyl laboratories, Montgomery, TX, USA), rabbit polyclonal anti-SOX2 (1:500 dilution; Millipore), and mouse monoclonal anti-β actin (1:2000 dilution; Sigma-Aldrich). Primary antibodies were detected with horseradish peroxidase (HRP)-linked antibodies: Goat anti-rabbit or goat anti-mouse (Santa Cruz Biotechnology, Dallas, TX, USA). Protein detection was performed using the NOVEX^® ECL system (Invitrogen, Carlsbad, CA, USA).

A SimpleChIP Enzymatic Chromatin IP Kit (Cell Signaling) was used for the chromatin immunoprecipitation (ChIP) assay. In detail, U251-MG cells harboring a doxycycline-inducible HA-PMLIV plasmid were grown in 150 mm dishes for 3 days and cross-linked with 35% formaldehyde for 10 min at RT. Then, cells were incubated for 5 min at RT after the addition of glycine, washed with PBS, and scraped into PBS with 100 µM phenylmethylsulfonyl fluoride (PMSF). Pelleted cells were lysed and nuclei harvested. Nuclear lysates were digested with micrococcal nuclease for 20 min at 37 °C and then sonicated in 500 μL aliquots on ice for 3 pulses of 15 s using a Branson sonicator. Lysates were clarified, and chromatin was stored at −80 °C until used. HA-Tag polyclonal antibody (Cell Signaling), rabbit polyclonal anti-PML (Bethyl laboratories), and normal rabbit IgG polyclonal antibody (Cell Signaling) were incubated overnight at 4 °C with rotation. After that, protein G-conjugated magnetic beads were added and incubated for 2 h at 4 °C. Washes and elution of chromatin were later performed and DNA quantification was carried out using a Viia7 Real-Time PCR System (Applied Biosystems) with SYBR Green and primers that amplify the predicted PML binding region to SOX9 promoter (chr17:70117013-70117409). Primer sequences used were forward 5′-ccggaaacttttctttgcag-3′ and reverse 5′-cggcgagcacttaggaag-3′.