Nf κb p65

Manufactured by Santa Cruz Biotechnology

Sourced in United States, Canada, Germany, United Kingdom, Brazil

NF-κB p65 is a subunit of the nuclear factor-kappa B (NF-κB) transcription factor complex. NF-κB p65 plays a central role in the regulation of genes involved in immune and inflammatory responses.

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243 protocols using nf κb p65

Antibody Characterization for Protein Detection

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Commercially available antibodies were used to detect FK2 (Enzo Life Sciences, BML-PW8810), DDK tag (Origene, TA50011), HCV NS3 (TORDJI-22, Abcam), HCV core (ThermoFisher Scientific, MA1-080), HAV capsid (Commonwealth Serum Laboratories, K24F2), Dengue capsid (Dr. Tom Hobman, University of Alberta), STAT1 (Cell Signaling Technologies, CST9175), STAT2 (Santa Cruz, sc-476), Y701-phosphoSTAT1 (Cell Signaling Technologies, 9167S), Y690-phosphoSTAT2 (Cell Signaling Technologies, 88410S), PDLIM2 (Abcam, 246868), p65 NF-κB (Santa Cruz, sc-372), Actin (EMD Millipore, MAB1501), PARP-1 (BD Pharmingen, BD556362), Lamin (Zymed, 33–2000), B-tubulin (Abcam, AB6046), Anti-NS5a (gift from Charlie Rice, 9E10). For western blotting, goat anti-mouse IR Dye 800 (Licor, 926–32210) and goat anti-rabbit IR Dye 680 (Licor, 926–32221) were used. For immunofluorescence, Alexa Fluor 546 goat anti-mouse IgG (ThermoFisher Scientific, A11030), Alexa Fluor 488 goat anti-rabbit IgG, (ThermoFisher Scientific, A11008), or Alexa Fluor 647 goat anti-mouse IgG (ThermoFisher Scientific, A21236) were used.

Joyce M.A., Berry-Wynne K.M., dos Santos T., Addison W.R., McFarlane N., Hobman T, & Tyrrell D.L. (2019). HCV and flaviviruses hijack cellular mechanisms for nuclear STAT2 degradation: Up-regulation of PDLIM2 suppresses the innate immune response. PLoS Pathogens, 15(8), e1007949.

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Western Blot Analysis of T Cell Signaling

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A total of 5×10⁶ CD4⁺ T cells in each group were extracted from the cytosolic and nuclear fractions using cell lysis buffer. The supernatants were separated, and the protein concentration in the fractions was quantified using a Bradford quantitative protein assay kit (Applygen Technologies Inc., China). The total protein lysates (40 μg per each well) were loaded and subjected to 10-12% SDS-PAGE before being electro-transferred to polyvinylidene difluoride membranes (PVDF) at 100 V for 1 h. The membrane was blocked with 5% nonfat milk in Tris-buffered saline supplemented with 0.05% Tween 20 (pH 7.6) at 4°C for 2 h and incubated with anti-mouse and anti-human polyclonal antibodies, including antibodies against ERK, phospho-ERK, JNK, phospho-JNK, NFAT1, p65 (NF-κB), phospho-p65 (NF-κB), p38, phospho-p38 and β-actin (1:1,000, Santa Cruz Biotechnology, Santa Cruz, CA, USA and Cell Signaling, USA), overnight at 4°C. The membrane was incubated with horseradish peroxidase-conjugated anti-mouse and anti-rabbit secondary antibody (1:2,000, Santa Cruz Biotechnology, Santa Cruz, CA, USA) at room temperature for 2 h. After extensive washing, the blot was developed using an ECL chemiluminescent detection kit (TransGen Biotech Co. Ltd., China) according to the manufacturer's instructions. The X-ray films were scanned and quantified by Gel-Doc (Bio-red) using image analysis software.

Samivel R., Kim D.W., Son H.R., Rhee Y.H., Kim E.H., Kim J.H., Bae J.S., Chung Y.J., Chung P.S., Raz E, & Mo J.H. (2015). The role of TRPV1 in the CD4+ T cell-mediated inflammatory response of allergic rhinitis. Oncotarget, 7(1), 148-160.

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Myocardial Infarction Inflammation Markers

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Samples were obtained from the border zone either at day 3 in Experiment 1 or 1 hour in Experiment 2 after MI. Antibodies to p65 NF‐κB (Santa Cruz Biotechnology), NLRP3 (Santa‐Cruz Biotechnology, sc‐34408), cleaved IL‐1β (Cell Signaling Technology) and β‐actin (Santa Cruz Biotechnology) were used. Western blotting procedures were described previously.¹¹ Experiments were replicated three times and results expressed as the mean value.

Lee T., Harn H., Chiou T., Chuang M., Chen C., Chuang C., Lin P, & Lin S. (2020). Host pre‐conditioning improves human adipose–derived stem cell transplantation in ageing rats after myocardial infarction: Role of NLRP3 inflammasome. Journal of Cellular and Molecular Medicine, 24(21), 12272-12284.

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Comprehensive IHC Analysis of Cellular Markers

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An IHC assay was performed as previously described²¹ (link) for detection of SEPT7 and p65 (NF-κB) (Santa Cruz Biotechnology, Dallas, TX, USA) and Akt, p-Akt, Snail, E-cadherin, N-cadherin, Vimentin, Ki67, BCL2, MMP2, and Cyclin D1 (Abcam, Shanghai, P.R. China) expression.
HE staining was also routinely performed. The processed slides were observed under an optical microscope (Olympus, Japan).

Wang W., Hao Y., Zhang A., Yang W., Wei W., Wang G, & Jia Z. (2021). miR-19a/b promote EMT and proliferation in glioma cells via SEPT7-AKT-NF-κB pathway. Molecular Therapy Oncolytics, 20, 290-305.

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Evaluating Transcription Factor Dynamics in Airway Cells

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The expression of FoxO3 in 16HBE cells and PBECs and the expression of SIRT1 and NF‐κB in 16HBE cells exposed to the previously described stimuli were evaluated by Western blot analysis. To study SIRT1, FoxO3 and NF‐κB nuclear expression, the protein extracts were treated to separate the cytoplasmic and nuclear protein fractions using a commercial kit ‘NE‐PER Nuclear and Cytoplasmic Extraction Reagents’ following the manufacturer's directions (Thermo Scientific; Waltham, MA, USA). For Western blot analysis, the following antibodies were used: SIRT1 (sc‐15404), FoxO3 (sc‐9812), p65/NF‐κB (sc‐372), Lamin B1 (sc‐377000) from Santa Cruz Biotechnology (Dallas, TX, USA); and β‐actin (A5441—Sigma‐Aldrich, St. Louis, MO, USA). Data are expressed as densitometric arbitrary units by correction with the density of the bands obtained for β‐actin. The quality of nuclear and cytoplasmic protein separation was assessed by measuring Lamin B1.

Di Vincenzo S., Heijink I.H., Noordhoek J.A., Cipollina C., Siena L., Bruno A., Ferraro M., Postma D.S., Gjomarkaj M, & Pace E. (2018). SIRT1/FoxO3 axis alteration leads to aberrant immune responses in bronchial epithelial cells. Journal of Cellular and Molecular Medicine, 22(4), 2272-2282.

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Protein Expression Analysis by Western Blot

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Protein extracts were boiled for 5 min in SDS sample buffer, separated using SDS-PAGE, and transferred onto polyvinylidene difluoride membranes (Immobilon™-P, MerckMillipore, Billerica, MA, USA). The membranes were probed with the following primary antibodies: rabbit anti-MMP-2 (1:1000; Novus Biologicals, Littleton, CO, USA), mouse anti-MMP-9 (1:300; Abcam, Tokyo, Japan), Akt (1:1000; Santa Cruz, California, USA), pAkt (1:1000; Santa Cruz), p65 NF-κB (1:1000; Santa Cruz), IкB-α (1:1000; Santa Cruz), ERK1/2 (1:1000; Abcam, Cambridge, UK), pERK1/2 (1:1000; Abcam), p38 (1:1000; Abcam), p-p38 (1:1000; Abcam), JNK (1:1000; Abcam), pJNK (1:1000; Abcam), c-Fos (1:500; Santa Cruz), c-Jun (1:500; Santa Cruz), iNOS (1:1000; Santa Cruz), and mouse anti-β-actin (1:10,000; Merck). All immunoreactive proteins were detected using ECL™ Western Blotting Detection Reagent (GE Healthcare, Buckinghamshire, UK).

Kugo H., Sukketsiri W., Iwamoto K., Suihara S., Moriyama T, & Zaima N. (2021). Low glucose and serum levels cause an increased inflammatory factor in 3T3-L1 cell through Akt, MAPKs and NF-кB activation. Adipocyte, 10(1), 232-241.

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Whole-cell Western Blot Analysis

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Whole‐cell extracts (WCEs) were prepared, and western analysis was performed as described [9 (link), 40 (link)]. The primary antibodies were the following: JAK1 (Proteintech, Rosemont, IL, USA, #66466‐I‐Ig; dilution 1 : 500), Bcl3 (Proteintech, #23959‐I‐AP; dilution 1 : 500), STAT1 (Cell Signaling, Danvers, MA, USA, #9172; dilution 1 : 1000), Ser727 p‐STAT1 (Cell Signaling, #8826; dilution 1 : 1000), p65 NFκB (Santa Cruz, #sc‐8008; dilution 1 : 200), K314/315 ac‐p65 (Signalway Antibody, Greenbelt, MD, USA, HW136; dilution 1 : 200), histone H3 (Abcam, Cambridge, MA, USA, Ab1791; dilution 1 : 1000), Lys9 acetyl histone H3 (Cell Signaling, #9649S; dilution 1 : 1000), and control actin (Sigma, St. Louis, MO, USA, A5060; dilution 1 : 2000).

Gaire B., Padmanabhan S., Zou Y., Uddin M.M., Reddy S.U, & Vancurova I. (2023). IFNγ induces Bcl3 expression by JAK1/STAT1/p65 signaling, resulting in increased IL‐8 expression in ovarian cancer cells. FEBS Open Bio, 13(8), 1495-1506.

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Protein Expression Analysis by Western Blot

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Western blotting was performed in cultured cells following various treatments. The protein lysates were measured by BCA assay and the same amount of proteins was resolved on SDS-PAGE followed by an electric transfer to a PVDF membrane. The blots after blocking were incubated with primary antibodies, including DKK1 (Abcam, Cambridge, MA, USA), p65 NF-κB (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and GAPDH (Santa Cruz Biotechnology). The blots were then incubated by HRP conjugated secondary antibody, and signals were developed by ECL-based imaging system.

Sun J., Chen J., Cao J., Li T., Zhuang S, & Jiang X. (2016). IL-1β-stimulated β-catenin up-regulation promotes angiogenesis in human lung-derived mesenchymal stromal cells through a NF-κB-dependent microRNA-433 induction. Oncotarget, 7(37), 59429-59440.

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Western Blot Analysis of Endothelial Cells

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EC were cultured in 0.2% FBS EBM-2 overnight before being lysed. Proteins were subjected to SDS-PAGE and immunoblotted with specific antibodies against Nox4 (provided by Dr. Ajah Shah), phospho-eNOS (p-eNOS, Ser1177, BD), phospho-p65 NFκB (Cell Signaling Technologies, CST), p65 NFκB (CST), VCAM1 (Santa Cruz), sEH (Santa Cruz); GAPDH (CST) was used as loading control. Proteins were visualized with an ECL system (Amersham Biosciences). Densitometry analysis was performed using NIH Image J software, and protein expression levels were normalized to GAPDH.

Hu P., Wu X., Khandelwal A.R., Yu W., Xu Z., Chen L., Yang J., Weisbrod R.M., Lee K.S., Seta F., Hammock B.D., Cohen R.A., Zeng C, & Tong X. (2017). Endothelial Nox4-based NADPH oxidase regulates atherosclerosis via soluble epoxide hydrolase. Biochimica et biophysica acta, 1863(6), 1382-1391.

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Protein Fractionation and Western Blot Analysis

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Total protein and mitochondrial and cytosolic protein fractions were isolated from mouse liver and western blotting performed, as previously described.(20 (link),21 (link)) Membranes were probed with the following antibodies: caspase 3 (Cell Signaling, Beverly, MA; #9665), caspase 7 (Cell Signaling; #9492), poly (ADP-ribose) polymerase (PARP; Cell Signaling; #9542), tubulin (Cell Signaling; #2148), cytochrome c (BD Biosciences, San Jose, CA; #556433), cytochrome oxidase (Abcam, Cambridge, MA; #14744), caspase 8 (R&D Systems; #AF1650), p50 NF-κB (Santa Cruz Biotechnology, Dallas, TX; #SC-114), p65 NF-κB (Santa Cruz; #SC-109), lamin A/C (Cell Signaling; #2032), cIAP1 (Enzo Life Sciences, Farmingdale, NY; #ALX-803-335-C100), cIAP2 (Santa Cruz; #SC-7944), superoxide dismutase 2 (SOD2) (Enzo Life Sciences; #ADI-SOD-111-D), Bcl-X_L (Cell Signaling; #2764), phospho-c-Jun N-terminal kinase (JNK; Cell Signaling; #9252), phospho-JNK (Cell Signaling; #4671) and Bid (kind gift of Xiao-Ming Yin, University of Indiana).

Zhao E., Ilyas G., Cingolani F., Choi J.H., Ravenelle F., Tanaka K.E, & Czaja M.J. (2017). Pentamidine Blocks Hepatotoxic Injury in Mice. Hepatology (Baltimore, Md.), 66(3), 922-935.

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