P nf κb

Mog (HPLC > 98%) was obtained from Chengdu Pufei De Biotech Co., Ltd. (Chengdu, China; CAS:88901-36-4). The compound was dissolved in dimethyl sulfoxide (DMSO, 0.1% final concentration in cultural medium). DMSO, LPS (Escherichia coli Serotype 055:B5), and LY294002 (10 μM, AKT inhibitor) were purchased from Sigma Chemical Co. (St Louis, USA). 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and phosphate-buffered saline (PBS) were supplied by Coolaber (Beijing, China). BCA were obtained from Beyotime Biotechnology (Shanghai, China). ECL chemiluminescence substrates were supplied from Millipore Corporation (Billerica, USA). COX-2, iNOS, TLR4, Nrf2 HMGB1, IL-1β, IL-18, NF-κB, p-NF-κB, MyD88, p-AKT, AKT, p-AMPK, AMPK, IL-6, and TNF-α were obtained from ABclonal Technology (Wuhan, China). Rabbit polyclonal antibodies against ERK (#4695), p-ERK (#4370), JNK (#9252), p-JNK (#4668), p38 MAPK (#8690), p-p38 (#4511), IκBα (#4814), and p-IκBα (#2859) were purchased from Cell Signaling Technology (Bossdun, USA). NQO1 (ab80588), HO-1 (ab68477), GCLC (ab207777), and GCLM (ab126704) were provided by ABCAm (Cambridge, United Kingdom). Dulbecco's modified Eagle's medium (DMEM) and trypsin were provided by Gibco (USA). Fetal bovine serum (FBS) was from Biological Industries (Uruguay, South America).

Experimental monolayers were washed with serum free media, and then total and fractionated proteins were extracted by cell lysis buffer (Cell Signaling Technology, Danvers, MA). The lysates were centrifuged at 12,000×g for 20 min at 4 °C. Equal amounts of protein, after concentration was determined by the Bradford assay (Bio-Rad, Hercules, CA), were loaded on SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad). After blocking, specific antibodies such as Bax, caspase-3, caspase-8, Bcl-2, PI3K, Notch1, p-NF-κB, NF-κB, E-cadherin, N-cadherin and β-actin from AB clonal Biotechnology Co., Ltd. (Wuhan, China) were used to perform detection. Finally each protein was detected using an enhanced chemilumi-nescence system (GE Healthcare, USA). Blot images were digitized (Chemidoc, Bio-Rad, Milan, Italy) and the area of each band was quantified using the computerized imaging system (QuantityOne, Bio-Rad). Relative optical density (arbitrary units) was normalized for control bands in each series and for protein loading (as probed by anti-actin blots). Each test was performed in triplicate experiments.

Brain tissues were collected and lysed in RIPA buffer in a mixture containing protease and phosphatase inhibitors (Beyotime Biotechnology, Jiangsu, China). Protein concentrations in each sample were calculated using the Bradford assay. Equal amounts of total protein from the tissue lysates were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride membranes (PVDF, Roche). The membranes were blocked in 5% dry milk (diluted with Tris buffered saline with Tween 20 (TBST) buffer) for 2 hours at room temperature and then incubated with rabbit polyclonal anti-iNOS, eNOS, p-eNOS (1 : 1,000; Cell Signaling Technology, USA), p-NF-κB (1 : 1000; ABclonal, China), and mouse monoclonal anti-β-actin (1 : 3,000; Boster Biological Technology, China) primary antibodies (diluted in 1X TBST with 5% bovine serum albumin (BSA)) for 16 hours at 4°C. The membranes were washed three times for 5 minutes with TBST at room temperature and incubated with a secondary horseradish peroxidase-conjugated sheep anti-rabbit or anti-mouse IgG antibody (1 : 5,000; Boster Biological Technology, CHINA) for 1 hour at room temperature. After washing three times for 5 minutes with TBST at room temperature, the protein bands were detected using horseradish peroxidase (Santa Cruz Biotechnology) and visualized using enhanced chemiluminescence.