Coimmunoprecipitation assay

Coimmunoprecipitation assays (co-IP) performed with uncrosslinked cells were grown to confluency in six-well plates. Cells were harvested and lysed in co-IP lysis/wash buffer (25 mM Tris-HCl pH 7.4, 200 mM NaCl, 1 mM EDTA, 0.5% NP40, 5% glycerol). Protein A/G agarose beads (EMD Millipore, cat. no. IP05) were incubated with primary antibody for 2 h at 4°C before addition to cell lysate. Lysate was incubated with beads-antibody complex overnight with rotation at 4°C. Beads were washed five times in co-IP lysis/wash buffer, resuspended in Novex NuPage Sample Loading Buffer (Fisher Scientific, cat. no. np0008) with 5 mM dithiothreitol (DTT) and boiled for 5 min at 95°C. Beads were then centrifuged at 8000 rpm and eluted protein removed with the supernatant and detected by western blot.

For crosslinked co-IP assays, cells were harvested from confluent 150-mm dishes. Cells were crosslinked using 1% formaldehyde for 15 min and then quenched with glycine (125 mM). Cells were washed in phosphate-buffered saline (PBS) and lysed in Buffer B (1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8.0) supplemented with protease inhibitors. Lysate was sonicated using a Bioruptor Pico (Diagenode) for 30 min and then centrifuged at maximum speed (20,000g) for 30 min at 4°C. Crosslinked lysate was diluted 10-fold in IP lysis buffer (0.01% SDS, 1.1% Triton-X, 1.2 mM EDTA, 16.7 mM Tris-HCl pH 8.0, 167 mM NaCl) treated with protease inhibitors (Sigma-Aldrich, cat. no. P8340) and benzonase (Millipore-Sigma, cat. no. 70746). Lysate was then incubated with rotation overnight with primary antibody at 4°C. Antibody-bound complexes were immunoprecipitated with Novex DYNAL Dynabeads Protein G (Invitrogen, cat. no. 10-003-D) or protein A/G agarose beads (EMD Millipore, cat. no. IP05) for 2 h at room temperature. Beads were washed five times using IP lysis buffer and eluted in 3.6 M MgCl₂ and 20 mM 2-(N-morpholino)ethanesulfonic acid (MES, pH 6.5) for 30 min with agitation. IP samples were then assayed for proteins by ELISA.

IP experiments were performed with antibodies to EWSR1 (clone B-1, Santa Cruz Biotechnology, cat. no. 398318), FLI1 (Abcam, cat. no. 15289), RNA Pol II (clone CTD4H8, EMD Millipore, cat. no. 05-623), phospho S5 RNA Pol II (Abcam, cat. no. 5131), and V5 (Abcam, cat. no. 27671). Elutions were probed by western assay for uncrosslinked samples or by ELISA for crosslinked samples.