Fluorescently tagged secondary antibodies

Cells were washed and incubated in lysis buffer [20 mM Tris–HCl pH 7.4, 100 mM EDTA, 10 mM NaCl, 1% Triton X-100, 1 mM β-glycerophosphate, 1 mM EGTA, 5 mM sodium pyrophosphate, ‘Complete’ protease inhibitor (Roche)] for 30 min on ice. Insoluble material was removed by centrifugation and protein concentration determined by Coomassie Plus protein assay (Thermo Scientific). Proteins were separated by gel electrophoresis and stained with silver nitrate or blotted for western analysis. Primary antibody binding was detected by fluorescently tagged secondary antibodies (Licor) and gel loading normalised using anti-actin or anti-Pol II antibodies (Abcam).

Mice were perfused with phosphate-buffered saline (PBS) and the tissues were dissected and snap-frozen with liquid nitrogen and kept at −80°C. On the day of the experiment, the frozen tissues were thawed and homogenized on the ice with bead homogenizer (Moni International) in ice-cold RIPA buffer [150 mM NaCl, 50 mM Tris–HCl (pH 8.0), 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate] with 1 mM phenylmethylsulfonyl fluoride, and 1× protease inhibitors (Roche). After centrifugation at 14 000 × g for 15 min at 4°C, supernatants were collected. Protein concentrations were determined via BCA assay, and then standardized. Samples were separated by 4–12% Bis-Tris PAGE (Invitrogen) and transferred to 0.2 µm nitrocellulose. Membranes were blocked with LiCor Odyssey blocking buffer for 2 h at room temperature followed by incubation with primary antibody overnight at 4°C with gentle rocking. Membranes were then washed with Tris-buffered saline with 0.1% Tween-20 (TBST) three times, 10 min each, and incubated with fluorescently tagged secondary antibodies (LI-COR Biosciences) for 1 h at room temperature, followed by three washes. Membranes were scanned using an Odyssey Infrared Imaging System (LI-COR Biosciences). Densitometry was performed using Image Studio (LI-COR Biosciences) and Image J.