BG4 IP

Immunoprecipitation was performed as described in⁸² with minor changes. 1 × 10⁶cells were seeded in 15 cm dishes. 24 h post seeding cells were irradiated with 10 J m⁻² UV light. Cells were crosslinked with a 4% PFA solution for 10 min at RT with gentle rocking followed by 10 min incubation with 0.125 mM glycine. Nuclei were isolated and lysed with the Chromatrap Hypotonic and Lysis buffer and DNA was sheared using a E220 Focused-Ultrasonicator. To the sheared chromatin 10 µg of BG4 antibody was added and incubated for 2 h at 4 °C under rotation. 2 µl of mouse monoclonal antibody (Sigma) against the DYKDDDDK epitope of BG4 was added to the solution and kept for 1 h at 4 °C under rotation. Samples were incubated with 50 µl Dynabeads-Protein G (Thermo Fisher Scientific) for 2 h at 4 °C. After washing two times with washing buffer (100 mM KCl, 0.1% (v/v) Tween-20 and 10 mM Tris-HCl pH 7.4) immunoprecipitated proteins bound to DNA were de-crosslinking by boiling the samples 10 min at 95 °C in 1× Laemmli buffer. The samples were then separated by SDS-PAGE and immunoblotted (as describe below).