Anti p nf κb

Total proteins were extracted from cells and tissues. The cells and tissues were lysed with 20 μL of RIPA Buffer (Roche Molecular Biochemicals, Switzerland), and protein concentrations were detected using the BCA kit (Beyotime, China), according to the manufacturer’s guidelines. Protein samples (20~40 μg) were separated on an SDS-PAGE gel and transferred to nitrocellulose membranes. After blocking the membranes with 5% skim milk in phosphate-buffered saline (PBS) for 1 h at room temperature, the membranes were probed with anti-IL-21 (Abcam, UK), anti-claudin-5 (GeneTex, USA), anti-p-NF-κB (Santa Cruz Biotechnology, USA), anti-NF-κB (Santa Cruz Biotechnology, USA), anti-p-ERK1/2 (Proteintech, USA), anti-ERK1/2 (Proteintech, USA), anti-p-JNK (Santa Cruz Biotechnology, USA), anti-JNK (Proteintech, USA), anti-p-P38 (Santa Cruz Biotechnology, USA), anti-P38 (GeneTex, USA) and GAPDH (Zhongshanjinqiao, China) antibodies at 4°C overnight. The nitrocellulose membranes were washed with PBST (PBS containing 0.5% Tween 20) three times for 7 min each, which was followed by the incubation with a fluorescence-labeled secondary antibody (IRDye700/800 mouse and rabbit antibodies) (Santa Cruz Biotechnology, USA). Protein levels were determined using an Odyssey infrared scanning system (LI-COR Biosciences, USA), and the band intensities were quantified using ImageJ software.

The antibodies used in the Western blot and immunofluorescence studies were anti-Nrf2 (sc-722), anti-HO1 (sc-136,961), anti-synaptosomal-associated protein 23 (SNAP-23) (sc-374,215), anti-PSD-95 (sc-71,933), anti-Syntaxin (sc-12,736), anti-tumor necrosis factor-α (TNF-α) (sc-52,746), anti-PARP-1 (sc-8007), TLR4 (sc-16240), Synaptophysin (sc-17750), anti-interleukin (IL)-1β (sc-32,294), anti-Bax (sc-7480), anti-Bcl2 (sc-7382), anti-p-NF-κB (sc-136,548), anti-Iba-1 (sc-32,725), anti-Glial fibrillary acidic protein (GFAP; sc-33,673), and anti-β- actin (sc-47,778) (Santa Cruz Biotechnology, Dallas, TX, USA). In addition, the anti-Cleaved Caspase-3 (#9664) antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). For Syntaxin and β-actin, the antibodies were diluted in TBST (1:10000) (Santa Cruz Biotechnology, Dallas, TX, USA). Other primary antibodies were diluted in 1x TBST (1:1000), and Secondary anti-mouse HRP (Horseradish peroxidase) conjugated (Promega Ref# W402) and anti-rabbit HRP conjugated (Promega Ref# W401) were diluted 1:10,000 in 1× TBST and were purchased from Promega, (Fitchburg, WI, USA). TAK242, Resatorvid (CAS 243984-11-4), the specific inhibitor of TLR4. For the confocal microscopic studies, the secondary fluorescent antibodies used were goat anti-mouse (Ref# A11029) and goat anti-rabbit (Ref# 32732) diluted in 1× PBS.

For relative protein expression, we followed the same methodological protocols of western blot as our previously published data (Gim et al., 2013 (link)). The whole tissue extract was centrifuged and resulted supernatant was carefully isolated. Bicinchoninic acid (BCA) kit (Pierce, Rockford, IL, USA) was utilized to determine protein concentration according to the guidelines provided by the manufacturer. 30 µg per sample of protein were electrophoresed and were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After blocking, the membrane PVDF was incubated with primary antibodies for the whole night at the refrigerator. The next morning secondary antibodies were applied, and the ECL detection reagent was used for x-ray bands. The antibodies used include anti-TNF-α, anti-p-JNK, Anti-JNK, anti-COX2, anti-IL-1β, anti-NOS2, anti-nuclear factor erythroid 2-related factor 2 (Nrf2), anti-p-NF-κB, anti-heme oxygenase-1 (HO-1), anti-Trx, and anti-β-actin (Santa Cruz, Biotechnology, CA, USA). The anti-mitogen activated protein kinase phosphorylated-P38 (MAPKp-P38) and anti-total-P38 were purchased from (Cell Signaling Technology, CST), and were used at dilutions 1:1000.

Western blotting was performed as previously described (23 (link), 24 ). Briefly, proteins were extracted using RIPA lysis buffer (Beyotime Biotechnology, Beijing, China) containing phenylmethanesulfonyl fluoride (PMSF) (Roche, Basel, Switzerland), and a protein phosphatase inhibitor (Roche, Basel, Switzerland). Thirty micrograms of proteins were subjected to 10% SDS-PAGE and then transferred to a polyvinylidene difluoride (PVDF) membrane (Roche, Rotkreuz, Switzerland). The membrane was blocked with 5% (w/v) skim milk in TBST (20 mM Tris–HCl [pH 8.0], 150 mM NaCl, and 0.1% [v/v] Tween-20) for 2 h and incubated with anti-heparanase (polyclonal antibody #733), anti-iNOS (Santa Cruz Biotechnology, TX, USA), anti-p/NF-κB (Santa Cruz Biotechnology, TX, USA), anti-p/ERK1/2 (Santa Cruz Biotechnology, TX, USA), anti-phospho-p38 (Santa Cruz Biotechnology, TX, USA), anti-GAPHD (Zhongshan Golden Bridge Biotech, Beijing, China) or anti-β-actin (Zhongshan Golden Bridge Biotech, Beijing, China) as primary antibodies. The membrane was washed with TBST, incubated with horseradish peroxidase-conjugated antibody against mouse IgG (1 h at room temperature), and rinsed with TBST. Proteins were visualized with ECL Western Blotting Detection Reagents (GE Healthcare, NJ, USA) and images were analyzed with Quantity One 4.1 software (Bio-Rad). The experiments were repeated at least three times.