Nf κb p65 transcription factor assay kit

Nuclear protein was isolated from cardiac tissues using the Nuclear and Cytoplasmic Extraction Reagents Kit (Thermo Scientific™ NE-PER™, Pittsburgh, PA, USA). Nuclear factor-kappaB (NF-κB) DNA binding activity in nuclei was determined using the NF-κB p65 transcription factor assay kit according to the manufacturer's instructions (Cayman Chemical, Ann Arbor, Michigan, USA). Briefly, 10 μg nuclear extracts were added to the designated wells with complete transcription factor binding assay buffer (100 μl/well) and incubated overnight at 4°C. After five times washing with 200 μl wash buffer, 100 μl of 1:100 diluted NF-κB p65 antibody was added for 1 hr at room temperature. After washing, 100 μl of 1:100 diluted goat anti-rabbit HRP conjugate was added to each well. 45 min. later, 100 μl of transcription factor developing solution was added to each well and incubated for 15 min. without light. Finally, 100 μl of stop solution was added and absorbance was measured at 450 nm.

NF-κB transcriptional activity was determined using the NF-κB (p65) Transcription Factor Assay Kit (Cayman Chemical #10007889). Primary astrocytes treated with OMVs (2.5 µg/ml, 12 h) or TNF (10 ng/ml, 48 h) were incubated for 60 min with or without the IKK (IMD 0354 [53 (link)] or BMS 345541 inhibitors [54 (link)]). Nuclear extracts were obtained from treated cells as previously described [49 (link)]. A volume of 10 µl containing normalized nuclear protein extracts was added per well, and adsorbed p65 was quantified following the manufacturer’s instructions.

Nuclear proteins were extracted from the kidney cortical tissues with a Nuclear Extract Kit (Cayman chemical, Ann Arbor, MI, USA). NF-κB DNA-binding activity was measured by NF-κB (p65) Transcription Factor Assay Kit (Cayman chemical, Ann Arbor, MI, USA) according to the manufacturer’s instructions. MDA level and SOD activity were quantified in kidney cortical tissues using Cayman’ assay kit (Cayman Chemical, Ann Arbor, MI, USA).

The NF-κB (p65) transcription factor assay kit (Cayman Chemical Company, MI, USA) was used to measure NF-κB (p65) binding activity in the nuclear extracts. The proteins extracted from nuclear or cytoplasmic fractions were taken in equal quantities and then (1) divided using SDS-PAGE, (2) relocated to PVDF membranes (Millipore, MA, USA), and obstructed using 5% nonfat milk in a Tris-Tween buffered saline buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 0.1% Tween-20) over a 3-hour period. Prior to conducting the analysis with Alpha Imager 2200 (Alpha Innotech Corporation, CA, USA), overnight incubation at 4°C was carried out with the primary antibodies, whilst 60-minute incubation at room temperature was carried out with the HRP-conjugated secondary antibodies. Finally, electronic quantification and normalization of signal intensity to Lamin B protein abundance were performed.

Recombinant IL-1β were purchased from R&D Systems (Minneapolis, MN). Antibodies for p-TAK1^(Thr184/187), TAK1^(Ser412), p-IRAK4^{(Thr345/Ser346)}, p-IRAK1^(Thr209), p-TAB2^(Ser372), IL-1β, p-STAT3^(Tyr705), p-STAT3^(Ser727), p-SAPK/JNK^{(Thr183/Tyr185)}, and IκBα were purchased from Cell Signaling Technologies (Danvers, MA; Cat# 90C7, 9339S, D6D7, T209, 8155, D3U3E, 9145S, 9136S, 4671S and 4812S). β-Actin and IRAK1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA; sc-47778; H273). Lipopolysaccharide (LPS), takinib and Tofacitinib were purchased from Sigma (St. Louis, MO, USA). The STAT3 Transcription Factor Assay Kit (Cat: 601950), 5Z-7-oxozeaenol, and NF-κB (p65) Transcription Factor Assay Kit (Cat: 10007889) were purchased from Cayman Chemicals (Ann Arbor, MI, USA) and NG 25 trihydrochloride was purchased from Axon Medchem (Reston, VA, USA).

Defatted soy protein and fat-free casein were procured from Sakthi Sugars Limited, Coimbatore, India, and Sisco Research Laboratory, Mumbai, India, respectively. Insulin and glucose assay kits were obtained from Monobind Microwells Inc, CA, USA, and Agappe Diagnostics Pvt. Ltd, Kerala, India, respectively. Antibodies such as phospho (T183/Y185) JNK and phospho-IKKα/β (Ser176/180) and anti-IKKβ were purchased from Cell Signaling Technology, MA, USA. Anti-JNK was obtained from Santacruz Biotechnology, CA, USA. Enhanced chemiluminescence (ECL, Immobilon HRP Western Substrate) kit was purchased from Thermo Scientific, MA, USA. Nuclear extraction kit and NF-κB p65 transcription factor assay kit were bought from Cayman Chemical Company, MI, USA. Supersensitive polymer-horseradish peroxidase immunohistochemistry detection kit was purchased from Biogenex laboratories, San Ramon, CA, USA. Antibodies, namely, anti-3-NT and anti-4-HNE, were purchased from Invitrogen, USA, and Merck (Calbiochem), Darmstadt, Germany, respectively. Primers were purchased from Sigma-Aldrich, MO, USA, and the SYBR Green-qPCR master mix was purchased from Thermo Scientific, MA, USA.

GM sulfate was obtained from Abbott Healthcare PVT Ltd, India. 2-thiobarbituric acid (TBA), antibodies against NF-κB (p65), cleaved caspase-3, Bax, Bcl-2, β-actin, and HRP-conjugated secondary antibody were purchased from Santa Cruz biotech (USA). NE-PER nuclear and cytoplasmic extraction kit was obtained from Pierce Biotechnology, Rockford, IL, (USA). NF-κB (p65) transcription factor assay kit was obtained from Cayman Chemical Company, Ann Arbor, MI. Rat TNF-α and IL-6 ELISA kits were obtained from R&D Systems, Inc. (USA). All other chemicals were of analytical grade.