Dynabeads

The immunoprecipitated proteins were eluted from the DynaBeads, separated on SDS-PAGE, and then transferred onto 0.2 μm nitrocellulose membrane (Bio-Rad). After incubating 1 h with blocking solution (PBS-T containing 5% nonfat milk), the membrane was probed overnight at 4°C with either rabbit polyclonal antibodies against KU70, KU86, PARP1 (Santa Cruz Biotechnology, SantaCruz, CA), or a mouse monoclonal antibody against DNA-PKc (Oncogene Research Products, Cambridge, MA). The antibody against GFP (or eYFP) was purchased from BD Biosciences (Palo Alto, CA). After washing with PBS-T, species-specific horseradish peroxidase-conjugated secondary antibody was added for 2 h at room temperature. Signals were generated with Western Lightning Chemiluminescence reagent plus kit (GE Healthcare Limited, Piscataway, NJ).

HEK293T cells were transfected with σNS expression plasmids alone or in combination with μNS expression plasmid using FuGene 6 transfection reagent (Promega) at a reagent/DNA ratio of 3:1 in Opti-MEM (Life Technologies). At 24 h posttransfection, cells were lysed with IP lysis buffer (25 mM Tris [pH 7.4], 150 mM NaCl, 1% NP-40 substitute [VWR], 0.5% deoxycholate [DOC], 0.1% SDS) on ice for 30 min or co-IP buffer (20 mM Tris [pH 7.5], 137 mM NaCl, 2 mM EDTA, 0.1% NP-40 substitute) at 4°C for 30 min with rotation. Lysis buffers were supplemented with protease inhibitors (Roche, 11873580001) before use. Following lysis, cellular debris was collected by centrifugation at 20,000 × g at 4°C for 20 min. Supernatants were incubated with protein G Dynabeads (Thermo Fisher, 10004D) saturated with σNS-specific monoclonal 2A9 (IP [10 (link)]) or 3E10 (co-IP [10 (link)]) antibodies at 4°C for 4 h with rotation. Antibodies were saturated on Dynabeads according to the manufacturer’s protocol. Dynabeads were washed with cold lysis buffer, and bound proteins were eluted by boiling in SDS-PAGE sample buffer (Bio-Rad) with β-mercaptoethanol for 10 min. Proteins were analyzed by immunoblotting.

Tosylactivated M‐280 dynabeads (ThermoFisher Scientific, Basingstoke, UK) were coated with antiserum for the E. coli O55 lipopolysaccharide (BioRad, Hercules, CA, USA) according to published methods (dynabeads_m280tosylactivated_man.pdf">https://tools.thermofisher.com/content/sfs/manuals/dynabeads_m280tosylactivated_man.pdf). Washed beads were coated with O55 antiserum for 14 h at 37°C in 0·04 mol l⁻¹ borate buffer pH 9·5, 1·2 mol l⁻¹ ammonium sulphate. Following overnight coupling, beads were washed in phosphate‐buffered saline (PBS)+0·5% bovine serum albumin (BSA), followed by two further washes in PBS + 0·1% BSA.