Mab5078

Cells were trypsinized, pelleted and washed twice with cold phosphate-buffered saline (PBS). Cell pellets were then lysed in cold low-ionic-strength buffer (50 mM NaCl, 1% IGEPAL, 10% glycerol, 10 mM HEPES, pH 7.4) with fresh added proteinase inhibitor cocktail, DTT and PMSF (ThermoFisher Scientific, San Jose, CA, USA). After the determination of protein concentration, samples were denatured in SDS sample buffer, sonicated three times for 15 s each time (Branson150) and centrifuged at 10,000 rpm for 20 min. Supernatants were heated at 95 °C for 5 min. Proteins were resolved using Criterion TGX 4–15% precast PAGE gels and transferred to nitrocellulose membranes with a Bio-Rad Trans-Blot Turbo transfer system. Primary antibodies used were anti-RelA (ab194726, Abcam, Waltham, MA, USA; MAB5078, R&D, Minneapolis, MN, USA); anti-actin (937215, R&D) was used as a loading control. Blots were imaged and quantified.

Anti-RelA (MAB5078, R&D, Minneapolis, MN, USA) and anti-OGG1 (NB100-106, Novus) antibodies (Ab) were used in PLA with Duolink PLA kit (DUO 92010, Millipore Sigma, ThermoFisher Scientific, San Jose, CA, USA) according to the manufacturer’s instructions. The nuclei were counter-stained with DAPI. The PLA signals were visualized in a fluorescence microscope (ECHO) at 20× magnification.