Anti phospho iκbα

Brazilin used in this study was isolated from the dried heartwood of Caesalpinia sappan L as described previously [30 (link)]. All commercial antibodies and chemicals were purchased from the following resources: Anti-IκB-α, anti-phospho- IκB-α and anti-phospho-p65 antibodies were from Cell Signaling Technology(Beverly, MA, USA); anti-LC3 (L7543), anti-actin(A2066) antibodies, bafilomycin A1 (Baf-A1; B1793), 3-methyladenine (3-MA; M9281), wortmannin (WM; W1628), tert-butyl hydroperoxide (t-BHP; 458139), chloroquine (CQ; C6628), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG; 70-25-7), bacterial LPS (from Escherichia coli, serotype 0127:B8) and Human pLKO.1 lentiviral contructs from Open Biosystem, targeted Atg5 specific shRNA (NM_004849) were from Sigma-Aldrich (St. Louis, MO, USA). A NF-κB-luc adenoviral vector (Ad5HSV- NF-κB-luc) was a gift from K.C. Sohn (Chungnam National University, Daejeon, Korea). Recombinant mouse TNFα was from R&D Systems (Minneapolis, MN, USA). The caspase inhibitor (z-VAD-FMK; 627610), N-acetyl-L-cysteine (NAC; 106425) and diphenyene iodonium (DPI) were from Calbiochem (San Diego, CA, USA). Mito-TEMPO [(2-(2,2,6,6 tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl) triphenylphosphonium chloride, monohydrate] (ALX-430–150-M005) and recombinant Fas Ligand (ALX-522-020-C005) were from Enzo Life Sciences (East Farmingdale, NY, USA);

Lung tissues were homogenized by a homogenizer and lysed using RIPA buffer containing a protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN). The protein concentration of each group was determined using BCA kits. Protein samples (30 μg) were separated by 10% SDS-polyacrylamide gel electrophoresis and subsequently transferred onto PVDF membranes, which were blocked by 5% skim milk for 1 hour at room temperature. After rinsing three times, the blots were incubated overnight at 4°C with the following primary antibodies: anti-TLR4 (1:1,000; Cell Signaling Technology), anti-NF-κB p65 (1:1,000; Cell Signaling Technology), anti-phospho-IκBα (1:1,000; Cell Signaling Technology), anti-IκBα (1:1,000; Santa Cruz), and anti-Cit-H3 (1:1000, Abcam). The membranes were washed three times, incubated with horseradish peroxidase–conjugated secondary antibodies (1:3,000; Sigma-Aldrich), and then visualized by an enhanced chemiluminescence kit (ECL plus). We used anti-GAPDH (1:2,000; Cell Signaling Technology), anti-β-actin (1:10,000; Sigma-Aldrich) and anti-Histone-H3 (1:1500; Sigma) as internal controls. To measure the relative ratio of protein expression, band intensities were quantified by Image-Lab software (Bio-Rad).

Phenyl Sepharose^TM High Performance and Q Sepharose^TM High Performance were supplied by GE Healthcare (Uppsala, Sweden). Fetal Bovine Serum (FBS) was from Sciencell (San Diego, CA, USA). Dulbecco’s modified eagle’s medium (DMEM) was from HyClone (Logan, UT, USA). The MMP assay kit with JC-1 and ROS Assay Kit were from Beyotime Institute of Biotechnology (Shanghai, China). Fluo-4 NW Calcium Assay Kits were supplied by Molecular Probes (Eugene, OR, USA). An Annexin V-FITC/propidium Iodide (PI) Apoptosis Detection Kit was from Nanjing KeyGen Biotech. Co., Ltd. (Nanjing, China). Anti-IκB-α and anti-p65 were from Easybio (Beijing, China). Anti-phospho-IκBα and anti-phospho-p65 were from Cell Signaling (Boston, MA, USA). Anti-beta-actin and ELISA kits were supplied by Yifeixue Bio Tech (Nanjing, China).

17-OHPC was synthesized by Symbiotec Pharmalab (Lot # ZHPCy19003) and provided by Evergreen Therapeutics, Inc. for the studies, castor oil (Sigma-Aldrich, #C9606) and 50% ethanol in PBS were used as the vehicles for in vivo and in vitro experiments, respectively. Muromonab-CD3 (OKT3) and anti-CD28 antibody were from Takara (#T210) and Sigma-Aldrich (# 217669), respectively. Phytohemagglutinin (PHA) was obtained from Sigma-Aldrich (# 11249738001). BrdU assay kit was from Cell Signaling Technology (# 6813). The antibodies used in this study included anti-phospho-IκBα (# 2859), anti-IκBα (# 9242), anti-NF-κB p65 (# 8242), anti-HDAC1 (# 5356) from Cell Signaling Technology, and anti-GAPDH (# ab9485) from Abcam. Unless otherwise specified, all cell culture reagents were purchased from Sigma-Aldrich or Life Technologies.

For western blot analysis, the cellular extracts were solubilized in 3× Laemmli buffer consisting of 1% SDS and boiled for 5 min at 95 °C. Equal protein amounts (30–80 μg) were separated on SDS–polyacrylamide gel electrophoresis (12.5–15% gels) and transferred to nitrocellulose membranes. Antibody detection was accomplished using the enhanced chemiluminescence method (Thermo Fisher Scientific, Darmstadt, Germany) and developed either with the Fusion SL Imager (Vilber Lourmat, Eberhardzell, Germany) or the Curix60 processor (Agfa healthcare, Bonn, Germany). The following antibodies were used in 3% milk/TBS–Tween (0.1%): anti-mFas (1:1000, Santa Cruz Biotechnology, Dallas, TX, USA), anti-actin (1:40,000, MP Biomedicals, Eschwege, Germany), anti-tubulin (Bio-Rad, Munich, Germany), anti-E-cadherin (BD Transduction laboratories, Heidelberg, Germany), anti-hFas, anti-p65, anti-phospho-p65, anti-IκBα, anti-phospho-IκBα, anti-cleaved caspase-3 and anti-Bcl-x_L (Cell Signaling, Leiden, The Netherlands).