Anti histone 3

To analyze the level of NF-kB, whole-cell, cytoplasmic, and nuclear proteins were extracted. Nuclear proteins were extracted using the NucBuster Protein Extraction kit (Novagen, Rockland, MA, USA). Briefly, 2!10 7 RAW264.7 cells were lysed with 150 ml of reagent 1 to remove cytoplasmic proteins. The pellet was resuspended in 1 ml of 100! Protease Inhibitor Cocktail, 1 ml of 100 mM DTT, and 75 ml of reagent 2. The nuclear protein extracts were collected using centrifugation at 16 000 g for 5 min at 4 8C.
A total of 40 mg of protein was mixed with 5!SDS-PAGE Sample loading buffer (Beyotime), boiled at 100 8C for 5 min. Proteins were transferred to PVDF membranes by electroblotting. The membranes were blocked using 5% non-fat dry milk in TBST for 1.5 h and then probed with anti-P50 (1:5000, Millipore, Billerica, MA, USA), anti-P65 (1:2000, Millipore), anti-IkBa (1:1000, Cell Signaling Technology, Danvers, MA, USA), anti-PIkBa (1:1000, Cell Signaling Technology), anti-histone 3 (1:5000, Millipore). Primary antibodies were detected using the DyLing TM800 Labeled Antibody to Rabbit/Mouse IgG (HtL) (1:10 000, KPL, Gaithersburg, MD, USA). Bound complexes were measured using the Odyssey Infrared Imaging System.