Nfκb p65

Jurkat cells were washed twice with ice-cold PBS. To extract cytosolic and nuclear protein, Nuclear and Cytoplasmic Protein Extraction Kit was used according to the manufacture's instructions (Beyotime, Haimen, China). Proteins from the extraction were resolved by electrophoresis, transferred to a PVDF membrane, and hybridized with antibodies for strep (GenScript, Nanjing, China, 1:2000), H3 (Abcam, Cambridge, UK, 1:4000), actin (GenScript, Nanjing, China, 1:2000), NFκB P65 (Sangon Biotech, Shanghai, China, 1:500), IKK and IκB (Beyotime, Shanghai, China, 1:1000). Next, the membranes were incubated in secondary antibody, IRDye® 800CW Goat anti-Mouse IgG and IRDye® 680RD Goat anti-Rabbit IgG (both from LI-COR Biosciences, Lincoln, NE) for 2 hours and the specific bands on the membranes were detected using Odyssey® Imaging Systems (LI-COR Biosciences, Lincoln, NE).

ADR was purchased from Shenzhen Main Luck Pharmaceuticals Inc. (Lot. 0901E); Urinalysis Reagent Strip from Acon Biotech (Hangzhou) Co., Ltd. (Lot. 201109144); Coomassie (Bradford) Protein Assay Kits from Nanjing Jiancheng Bioengineering Institute (Lot. 20120413 and 20120604); Interleukin-4 and Interleukin-8 ELISA kits for rats from Abcam (HK) Ltd. (Lot. ISC10330EIA-2121R and -2135-R, resp.), Rabbit Anti-Rat IgG/FITC from Beijing Biosynthesis Biotechnology Co., Ltd. (Lot. 990603), SOD and MDA Assay Kits from Nanjing Jiancheng Bioengineering Institute (Lot. 20120523 and 20120324); Trizol (9108, Lot. number AK8106), RT Reagent Kit (RR047A, Lot. AK2003), and SYBR Premix ExTaq II (RR820A, Lot. number AK4401) were from Takara, Dalian, China; the primers for NF-κB p65, IκB, and GAPDH were synthesized by Sangon Biotech (Shanghai) Co., Ltd.