Anti nf κb p65

HEY cells were cultured on 12-mm glass cell culture coverslips (Thermo Fisher Scientific). Cells were washed three times with phosphate-buffered saline (PBS) and then fixed for 15 min in PBS with 4% paraformaldehyde. After washing with gentle shaking, cells were permeabilized for 5 min with methanol and washed. The proximal-ligation assay to detect the interaction of p50 with p65, anti- NF-κB p50 (Santa Cruz Biotech, Dallas, TX; Cat: sc-8414) and anti-NF-κB p65 (Santa Cruz; Cat: sc-372) and for p65- SIRT1 interaction, anti-NF-κB p65 (Santa Cruz Biotech; Cat: sc-8008) and anti-SIRT1 (Santa Cruz; Cat: sc-15404) were used with a Duolink PLA assay kit (Sigma Aldrich). Images were acquired Zeiss LSM 700 (Zeiss).

ChIP assays were performed as described previously (28 (link), 29 (link)). Briefly, cells were fixed with 1% formaldehyde for 10 min, collected in ice-cold PBS, and resuspended in an SDS lysis buffer. Genomic DNA was then sheared to lengths ranging from 200 to 1000 bp by sonication. Cell extracts were incubated with either anti-NF-κB p65 (Santa Cruz, Dallas, TX), anti-H3K4me3 (Cell Signaling Technology, Danvers, MA), anti-RNA Polymerase 2 (Millipore, Burlington, MA), or normal mouse IgG (Santa Cruz, Dallas, TX) overnight at 4°C, followed by precipitation with protein G-agarose beads. The immunoprecipitates were sequentially washed once with a low-salt buffer, once with a high-salt buffer, once with an LiCl buffer, and twice with a Tris buffer. The DNA–protein complex was eluted, and proteins were then digested with proteinase K for 1 h at 45°C. The DNA was detected by qRT-PCR analysis as described above and analyzed using the fold enrichment method. Gene-specific PCR primer pairs are listed in Supplementary Table 1.

BMDCs (4 × 10⁶) isolated, differentiated and cultured as described above were aliquoted into 250 μl PRF HBSS in 1.5 ml screw-cap polypropylene tubes. Cells were starved for 2 h at 37 °C prior to stimulation by the addition of 250 μl 2×-concentrated stimuli at the appropriate time points. Stimulation was stopped with the addition of 1 ml ice-cold HBSS and transfer to wet ice. Cells were immediately centrifuged at 8000g for 1 min at 4 °C, snap-frozen on dry ice or liquid N₂, and stored at −80 °C until analysis.
Cells were lysed using RIPA buffer (50 mM Tris–HCl pH 7.4, 1% NP-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA). Lysates were clarified by centrifugation at 10,000g for 10 min and the supernatants transferred to new 1.5 ml tubes. Protein contents of the lysates were quantified using the BCA 96-well plate assay (ThermoFisher) according to the manufacturer’s instructions. Samples were fractionated on 4–12% gradient NuPAGE Bis–Tris precast polyacrylamide gels, followed by transfer to polyvinylidene difluoride (PVDF) membranes according to the manufacturer’s instructions (Invitrogen; ThermoFisher). Blots were probed with anti-phospho-NFκB p65 (Ser536), anti-vinculin, anti-β-actin (Cell Signaling), and anti-NFκB p65 (Santa Cruz) antibodies. Uncropped immunoblot scans are presented in Supplementary Fig. 9.