Western blotting—chromatin fractionation and antibodies

Cells were harvested and washed with PBS. To release soluble factors, the cell pellet was resuspended in ice-cold chromatin extraction buffer (20 mM HEPES (pH 7.9), 1.5 mM MgCl₂, 140 mM NaCl, 300 mM Sucrose, 0.5% TX-100, Complete EDTA-free Protease Inhibitor Cocktail (Merck), PhosSTOP phosphatase inhibitors (Merck) and 20 μM MG132 (Sigma Aldrich). The cell pellet was incubated in the extraction buffer for 5min at 4°C with gentle mixing (300 rpm), and soluble and chromatin bound fractions were separated by centrifugation. The chromatin bound pellet was washed once in extraction buffer, followed by chromatin digestion with 100 U/ml benzonase (Sigma Aldrich) in extraction buffer for 2h at 4°C with gentle mixing (300 rpm). Both soluble and chromatin bound fractions were added Lane Marker Reducing Sample Buffer (Pierce Biotechnologies) and boiled at 95°C prior to analysis by quantitative western blotting. The final volumes of chromatin bound and soluble fractions were kept equal to allow comparison of the two fractions. Criterion TGX Stain-free gels (BioRad) and nitrocellulose membranes (BioRad) were used for separation and transfer respectively. Criterion Stain-free imager was activated in a Chemidoc MP (BioRad) prior to transfer. Antibodies used were: total RNAPII (F-12, Santa Cruz Biotechnologies), pRNAPII S5 (3E8) and pRNAPII S2 (3E10) (Sigma Aldrich). Total protein levels obtained from stain-free signal on membranes were used as loading control. Blots were imaged using chemiluminescence substrates (Supersignal west pico, dura or femto from Thermo Scientific). The Image Lab 4.1 (BioRad) software was used for quantifications and processing of images. Saturated signals were excluded. For accurate quantifications, a dilution curve of one of the samples was included. Membranes were stripped using ReBlot Plus Mild Antibody Stripping Solution (Millipore) in order to allow a new round of blotting for proteins.