Bs3 crosslinking agent

Cross-Linking experiments were performed as previously described^{12 (link)}. Briefly, serum starved SH-SH5Y cells were washed with ice-cold PBS then treated with resistin (200 ng/mL) for 1 h at 4 °C. BS3 crosslinking agent (Thermo Scientific, Illkirch, France) was then added directly to the incubation solution to a final concentration of 2.5 mM and followed by an additional incubation for 1 h at 4 °C. Crosslinking reaction was stopped by the addition of a quenching solution (20 mM Tris–HCL pH 7.5). Cells were solubilized and protein lysates were then subjected to immunoprecipitation using antibodies raised against TLR4. Western blots were performed as described before and membranes were immunoblotted with antibodies raised against TLR4 or resistin.

Magnetic Dynabeads™ protein G (Invitrogen) were first washed in PBS/0.01% Tween-20 (PBST) twice (30µL beads/condition). The beads were then incubated for 1 h at room temperature with 5 μg Flag or mouse IgG antibody in PBST. The antibody was crosslinked to the beads using BS₃ crosslinking agent (Thermofisher, #21580) according to the manufacturer’s protocol. The beads were then washed three times with PBST and equilibrated in 2 volumes/ diluted lysate of 1% NP-40 lysis buffer. One hundred micrograms of chromatin extracts were diluted 10 times in dilution buffer (50 mM Tris pH 8; 150 mM NaCl) and incubated with the crosslinked beads for 1.5 h at 4 °C. The beads were then washed twice in 1% NP-40 lysis buffer followed by two more washes in 0.2% NP-40 lysis buffer. The beads were eluted twice in 20 μL 0.1 M citric acid pH 2.6 for 5 min each, at room temperature with mild agitation. The eluates were neutralized in 5 μL of 2 M Tris pH 8.

Magnetic Dynabeads™ protein G (Invitrogen) were first washed in PBS/0.01% Tween-20 (PBST) twice (30 µL beads/condition). The beads were then incubated for 1 h at room temperature with 2 μg RNF168 or mouse IgG antibody in PBST. The antibody was crosslinked to the beads using BS₃ crosslinking agent (Thermofisher, #21580) according to the manufacturer’s protocol. The beads were then washed three times with PBST and equilibrated in 4 volumes/lysate of 1% NP-40 lysis buffer. 150–200 μg of nuclear extracts (combined soluble and insoluble nuclear fractions) were incubated with the crosslinked beads for 2 h at 4 °C. The beads were then washed once in 1% NP-40 lysis buffer, followed by three washes in 0.2% NP-40 lysis buffer. The beads were eluted twice in 20 μL 0.1 M citric acid pH 2.6 for 2 min each, at room temperature with mild agitation. The eluates were neutralized in 5 μL of 2 M Tris pH 8.