Igg mouse

Immunoprecipitation experiments were realized with 150 μg of total protein amounts from the cytosolic and nuclear fraction in normoxia or hypoxia, with a HA antibody (H9558/H3663, Sigma) 72 μg (2.4 mg/mL) or IgG mouse control (Sigma I5381) (2 mg/mL) using EZ view red protein G beads (Sigma). The beads-antibody-protein mix was incubated overnight at 4 °C and bounds protein were eluted with 35 μg HA peptides diluted in PBS (Sigma); then Laemmli buffer was added and the eluate heated at 95 °C 2 min.

One 15-cm plate containing cells grown to 80% confluence was used for each treatment condition and divided into aliquots for different immunoprecipitation conditions or antibodies. To harvest, cells were washed twice with PBS on the plate, then lysed on ice using a cell scraper with lysis buffer (150 mM KCl, 0.2% (v/v) NP-40, 10% (v/v) glycerol (v/v), 20 mM Tris pH 7.5, 0.5 mM DTT) containing freshly added protease inhibitor complex (Roche). Following 15 min incubation on ice, lysates were cleared at high speed centrifugation at 14,000 rpm for 10 min before protein quantitation using the BCA assay (Pierce). Equal amounts of lysate (500 μg– 1 mg) were immunoprecipitated while rotating at 4°C with 3 μg of one of the following antibodies: Anti-Flag M2 affinity Gel (Sigma), PP4-C and PP4R2 (Bethyl) or IgG controls: IgG-Mouse or IgG-Rabbit (Sigma). Protein G agarose beads (30 μl) were added to each immunoprecipitate and rotated overnight. Beads were washed four times with lysis buffer, and then resuspended in SDS loading dye and boiled prior to immunoblotting.

Ishikawa and Hec50 cells were grown in stripped media for 24 h in T25 flask. SAHA treatments at 2.5 μM were added for 48 h. Following that time, approximatively 5 × 10⁶ cells for each cell line were processed for ChIP analyses following manufacturer’s instructions (Chromatrap^® ChIP-seq, Norfolk, UK) [67 ]. The following antibodies were used; Active Motif, AcH3 (#39140, ChIP-seq); Sigma-Aldrich, IgG rabbit (#12-370, ChIP-seq), IgG mouse (#12-371, ChIP). PCR was used as the endpoint assay to analyze the ChIP DNA, using a BioRad CFX real time PCR machine (BioRad, Watford, UK). The primers sequences used were P21 Forward 5′-CCCACAGCAGAGGAGAAAGAA-3′, P21 Reverse 5′-CTGGAAATCTCTGCCAGACA-3′; P53 Forward 5′-AGACCAGCCTGACCAATA-3′, P53 Reverse 5′-GCTCCCGGAAACATCTCAC-3′. The human GAPDH primers present in the Chromatrap^® ChIP-seq kit was used as a positive control. The ratios of the signals in immunoprecipitated DNA versus input DNA was calculated as previously shown [68 (link)].