P nf κb

Manufactured by Cell Signaling Technology

Sourced in United States, China, United Kingdom

P-NF-κB is an antibody that specifically detects the phosphorylated form of the NF-κB p65 subunit. NF-κB is a transcription factor that plays a key role in regulating the immune response, cell survival, and inflammation.

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P-NF-κB p65 rabbit monoclonal PE-conjugated antibodies to p-NF-κB P-NF-κB p65 Phospho-NF-κB p65 (p-NF-κB p65) Antibodies against p-NF-κB p65 P-NF-κB-Ser536 Anti-phospho-NF-κB p65 (Ser536) (p-p65) Anti-p-NF-κB (Ser 536) antibody P-NF-κB antibody Phosphorylated NF-κB (p-NF-κB;

192 protocols using p nf κb

Cardiac Protein Analysis via Western Blotting

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For the Western blotting assay, frozen heart tissues were lysed as previously described [21 (link),22 (link)]. After the loading concentration was normalized at 50 μg per well, the proteins were separated by 8–12% SDS-PAGE and then transferred onto PVDF membranes (Merck Millipore). Next, the membranes were blocked by 5% skim milk powder dissolved in Tris-buffered saline containing 0.1% Tween-20, washed 3 times, and probed with corresponding primary antibodies including MD-1 (1: 800, Santa Cruz, USA), TLR4 (1: 600, Santa Cruz, USA), p-P38MAPK (1: 500, Cell Signaling, Danvers, MA), p-ERK1/2 (1: 700, Cell Signaling, Danvers, MA), p-JNK (1: 1000, Cell Signaling, Danvers, MA), p-NF-κB (1: 500, Cell Signaling, Danvers, MA), P38MAPK (1: 600, Cell Signaling, Danvers, MA), ERK1/2 (1: 900, Cell Signaling, Danvers, MA), JNK (1: 600, Cell Signaling, Danvers, MA), p-NF-κB (1: 800, Cell Signaling, Danvers, MA), and NF-κB (1: 800, Cell Signaling, Danvers, MA) at the recommended dilution overnight at 4°C. After incubation with horseradish peroxidase-conjugated secondary antibodies for 60 min, the protein bands were visualized with enhanced chemiluminescence reagent. GAPDH was used as a loading control for whole cellular protein.

Zhang Y.J., Huang H., Liu Y., Kong B, & Wang G. (2019). MD-1 Deficiency Accelerates Myocardial Inflammation and Apoptosis in Doxorubicin-Induced Cardiotoxicity by Activating the TLR4/MAPKs/Nuclear Factor kappa B (NF-κB) Signaling Pathway. Medical Science Monitor : International Medical Journal of Experimental and Clinical Research, 25, 7898-7907.

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Alzheimer's Disease Signaling Pathway

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rhIL-1β, rmIL-1β, forskolin, and the inhibitors NS398, LY294002, H89, and KT5720 and an antibody specific for mPGES-1 were obtained from Sigma-Aldrich Corp (St. Louis, MO, USA). The antibody specific for BACE-1 and the inhibitor specific for HSP70, VER155008 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against β-actin, COX-2, p65, Aβ, Akt, p-Akt (Ser 473), CREB, p-CREB (Ser 133), p-NF-κB (Ser 536), p-NF-κB (Ser 276), NF-κB, and IL-1β and siRNAs specific for AKT and CREB were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). p65, mPGES-1 cDNA, and the empty pCMV6-XL vector were obtained from Origene Technologies (Beijing, China). The PGE₂ and cAMP enzyme immunoassay kits were from Cayman Chemical (Ann Arbor, MI, USA). IL-1β enzyme immunoassay kits were obtained from Raybiotech, Inc. (Norcross, GA, USA). All reagents for the qRT–PCR and SDS-PAGE experiments were purchased from Bio-Rad Laboratories (Hercules, CA, USA). All other reagents were from Invitrogen (Carlsbad, CA, USA) unless otherwise specified.

Wang P., Guan P.P., Wang T., Yu X., Guo J.J, & Wang Z.Y. (2014). Aggravation of Alzheimer’s disease due to the COX-2-mediated reciprocal regulation of IL-1β and Aβ between glial and neuron cells. Aging Cell, 13(4), 605-615.

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Apoptosis Signaling Pathway Analysis

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Cells were treated with doxycycline at 1 g/ml, with 15 nM Actinomycin D (ActD, Invitrogen) for the indicated times in each case. Total protein from MCF7ras, HCC1806 or HS578t were obtained using Pierce RIPA buffer reagent (ThermoFisher), including a cocktail of proteases inhibitors and phosphatases inhibitors (Roche). 20 µg of protein were loaded into SDS-PAGE gels with a gradient from 4 to 15% (Bio-Rad). Proteins were transblotted in a wet-transfer system (Bio-Rad) to PVDF membranes (Immobilon-P, Millipore). Next, membranes were blocked with 5% non-fat milk in TBS and 0,01% Tween-20 (v/v). Membranes were blotted with antibodies against Bim, Bax, Bcl2, Bad, Bid, Puma, PARP, cleaved PARP, caspase 7, cleaved caspase 7, caspase 9, cleaved caspase 9, EGFR, HER2, HER3, AKT, pAKT 473, p42 p44 MAPK, pMEK1/2, LRP6, P-LRP6, Wnt5 a/b, Axin, NF-κB, pNF-κB, IKKα, IKKβ, IκBα, GAPDH (1:1000 from Cell Signaling) and LACTB and Caspase 8 (1:1000, ProteinTech). Signal was detected with enhanced chemiluminescence (ECL) using Azure c600 Western blot Imaging system.

Gonzalez-Morena J.M., Escudeiro-Lopes S., Ferreira-Mendes J.M., Jakoube P., Cutano V., Vinaixa-Forner J., Kralova Viziova P., Hartmanova A., Sedlacek R., Machado S., Malcekova B, & Keckesova Z. (2022). LACTB induces cancer cell death through the activation of the intrinsic caspase-independent pathway in breast cancer. Apoptosis, 28(1-2), 186-198.

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Western Blot Analysis of Protein Biomarkers

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Cells were lysed with 10 μL/mL Radio-Immunoprecipitation assay Lysis Buffer containing protease inhibitors (Beyotime), centrifuged at 14,000 × g, and quantified by bicinchoninic acid protein assay (Beyotime). Total protein (20 μg) was loaded into a sodium lauryl sulfate polyacrylamide gel and separated. Then, the sample was electroblotted onto a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA), blocked with 5% skim milk, and incubated with the following primary antibodies: β-actin (A5441, MilliporeSigma), KLF6 (14716-1-AP, Proteintech), Nrf2 (16396-1-AP, Proteintech), NF-κB (14220-1-AP, Proteintech), p-NF-κB (8242, Cell Signaling Technology), HO-1 (10701-1-AP, Proteintech), Bax (50599-2-Ig, Proteintech), Bcl-2 (12789-1-AP, Proteintech), and Ki-67 (27309-1-AP, Proteintech). The membrane was then incubated with the secondary antibody conjugated with horseradish peroxidase (Beyotime), developed by enhanced chemiluminescence kit (Amersham Pharmacia Biotech, Little Chalfont, UK) and imaged by ChemiDoc XRS imaging system. Image J image analysis software was used for data analysis. Three independent replicates were performed for each group.

Zhou S., Li J., Zhang X, & Xiong W. (2022). MicroRNA-124 modulates neuroinflammation in acute methanol poisoning rats via targeting Krüppel-like factor-6. Bioengineered, 13(5), 13507-13519.

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Cytotoxicity Evaluation of Compounds

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Dulbecco’s modified Eagle’s medium (DMEM), Hoechst 33,342, Fluoromount, 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA), and Rhodamine-123 and Fetal Bovine Serum (FBS) were purchased from Sigma (St. Louis, MO, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and trypsin were procured from Hi-media Pvt. Limited, Mumbai (India). Rabbit monoclonal Bcl-xl, p-53, p-NF-κB, Caspase3, and Caspase9 antibodies and anti-rabbit- HRP secondary antibody were obtained from Cell Signaling Technology, Danvers, MA, USA. PVDF membrane (MDI, Ambala). The RT-PCR chemical kit was purchased from Bio-Rad, CA, USA. The BD Cycletest plus DNA Kit was from BD Biosciences, San Jose, CA, USA. Chemicals and reagents of analytical (AR) grade were used to perform the experiments.

Kumar A., Kaur S., Dhiman S., Singh P.P., Bhatia G., Thakur S., Tuli H.S., Sharma U., Kumar S., Almutary A.G., Alnuqaydan A.M., Hussain A., Haque S., Dhama K, & Kaur S. (2022). Targeting Akt/NF-κB/p53 Pathway and Apoptosis Inducing Potential of 1,2-Benzenedicarboxylic Acid, Bis (2-Methyl Propyl) Ester Isolated from Onosma bracteata Wall. against Human Osteosarcoma (MG-63) Cells. Molecules, 27(11), 3478.

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Western Blot Analysis of Signaling Proteins

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Total proteins were extracted using cold RIPA (Millipore) containing a protease inhibitor. The protein concentration was centrifuged to remove the pellet and debris. Total proteins were separated by 10% SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membranes (Millipore) and then blocked with 5% fat-free milk. Expressions of NF-κB, P- NF-κB, ERK1/2, and P-ERK1/2 (Cell Signaling) and GAPDH (Abcam) proteins were determined using the respective specific antibodies (1/1000). After a membrane was incubated with a secondary antibody, it was then visualized using an enhanced chemiluminescence system (Bio-Rad).

Li D., Zhuo Y., Zhang Q., Zhang L., Zhang S., Lv Y., Li C., Cui L., Guan X., Yang L, & Wang X. (2019). Purification of 3, 4-dihydroxyphenylethyl alcohol glycoside from Sargentodoxa cuneata (Oliv.) Rehd. et Wils. and its protective effects against DSS-induced colitis. Scientific Reports, 9, 3222.

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Phloretin Modulates Inflammatory Pathways

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All chemicals, unless otherwise stated, were of the highest quality and were used as supplied. Phloretin (with ≥99% purity) was purchased from Sigma-Aldrich (USA). Purity of Phloretin was confirmed by HPLC and Mass spectrometer (KBSI). Anti- myeloid differentiation primary response 88 (MyD88), phosphorylation of transforming growth factor beta-activated kinase 1 (p-TAK1), antibodies were purchased from Abcam (UK). Anti-cyclooxygenase-2 (COX-2), hemeoxygenase-1 (HO-1), Actin, and p-NF-κB antibodies were purchased from Cell Signaling (USA).

Chauhan A.K., Jang M, & Kim Y. (2019). Phloretin Protects Macrophages from E. coli-Induced Inflammation through the TLR4 Signaling Pathway. Journal of Microbiology and Biotechnology, 30(3), 333-340.

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Investigating Inflammatory Signaling Pathways

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Recombinant IL-1β, forskolin and the inhibitors LY294002, U0126, SB203580 and SP600125 were obtained from Sigma-Aldrich Corp. Antibodies specific for β-actin, AKT, p-AKT (Ser 473), ERK1/2, p-ERK1/2 (Thr 202/Tyr 204), p38, p-p38 (Thr 180/Tyr 182), c-Jun, p-c-Jun (Ser 63), NF-κB, p-NF-κB (Ser 536), p-NF-κB (Ser 276), IL-1β and siRNAs targeting AKT, ERK1/2, p38 were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Quinazoline (QNZ) was obtained from Enzo Life Sciences (Farmingdale, NY, USA). The IL-1β and cAMP enzyme immunoassay kits were from Cayman Chemical (Ann Arbor, MI). Recombinant human MMP-7 was obtained from R&D systems (Minneapolis, MN, USA). MMP-7 cDNA plasmids were purchased from Origene Technologies (Rockville, MD, USA). All reagents for qRT-PCR and SDS-PAGE experiments were purchased from Bio-Rad Laboratories. All other reagents were from Invitrogen (Carlsbad, CA) unless otherwise specified.

Guan P.P., Yu X., Guo J.J., Wang Y., Wang T., Li J.Y., Konstantopoulos K., Wang Z.Y, & Wang P. (2015). By activating matrix metalloproteinase-7, shear stress promotes chondrosarcoma cell motility, invasion and lung colonization. Oncotarget, 6(11), 9140-9159.

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T cell Signaling Proteins Analysis

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T cells were purified from spleens and lymph nodes using EasySep CD4⁺ purification kits (STEMCELL Technologies). Equal numbers of T cells from WT, LAT^m/m, and IL6^−/−LAT^m/m mice were lysed, resolved on SDS-PAGE, and blotted with antibodies against the following proteins: Zap70, pLck, Lck, pERK, ERK2, pAkt (Ser473), Akt, pP38, P38, p NFκB, and NFκB (Cell Signaling).

O’Brien S.A., Zhu M, & Zhang W. (2015). The Importance of Interleukin-6 in the Development of LAT-mediated Autoimmunity. Journal of immunology (Baltimore, Md. : 1950), 195(2), 695-705.

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Mango Extract Effects on Cellular Pathways

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After a 24 h mango extract treatment, cells were lyzed in RIPA buffer, a 1% protease and proteinase inhibitor cocktail were added (Pierce/Thermo Scientific, Rockford, IL) and centrifuged [36 (link)]. Mucosal scrapings were homogenized in a buffer (500 mM Tris–HCL, 1M Sucrose, 200 mM EDTA, 100 mM EGTA, 0.4M NaF, 10% Triton X–100, 10 mM Sodium Orthovanadate, and Protease Inhibitor Cocktail) then centrifuged at 15,000g for 10 min at 4°C, and the supernatant was stored at −80°C. Fifty microgram of protein was loaded onto the gel, followed by transfer onto PVDF membrane. Membranes were incubated with primary antibodies against NF-κB, p-NF-κB, COX-2, cleaved caspase-3, PARP-1 (Cell Signaling Technology, Beverly, MA), iNOS (Cayman Chemical, Ann Arbor, MI), PI3K (p85β), HIF-1α (Abcam, Cambridge, MA), and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA).

Kim H., Banerjee N., Barnes R.C., Pfent C.M., Talcott S.T., Dashwood R.H, & Mertens-Talcott S.U. (2016). Mango Polyphenolics Reduce Inflammation in Intestinal Colitis—Involvement of the miR-126/PI3K/AKT/mTOR Axis In Vitro and In Vivo. Molecular carcinogenesis, 56(1), 197-207.

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