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Pnfκb p65 s536

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The PNFκB P65 S536 is a product from Cell Signaling Technology that is used to detect and quantify the phosphorylation of the p65 subunit of NF-κB at serine 536. It is a reagent intended for research use only.

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Lab products found in correlation

Nf κb p65, by Cell Signaling Technology (4 mentions) β actin, by Merck Group (3 mentions) Gapdh, by Cell Signaling Technology (2 mentions) β actin, by Cell Signaling Technology (2 mentions) Pgsk 3αs21βs9, by Cell Signaling Technology (1 mentions) Pikbαs32, by Cell Signaling Technology (1 mentions) Ikkα ikkβ, by Cell Signaling Technology (1 mentions) Pβ catenins33 37 t41, by Cell Signaling Technology (1 mentions) Ne per nuclear and cytoplasmic extraction kit, by Thermo Fisher Scientific (1 mentions) β catenin, by Cell Signaling Technology (1 mentions) Gsk3β, by Cell Signaling Technology (1 mentions) Pakt s473, by Cell Signaling Technology (1 mentions) P cjuns73, by Cell Signaling Technology (1 mentions) Ecf western blotting system, by Cytiva (1 mentions) Piκbαs32 36, by Cell Signaling Technology (1 mentions) Pikkα βs176 180, by Cell Signaling Technology (1 mentions) Cox 2, by Cayman Chemical (1 mentions) Dc protein assay kit, by Bio-Rad (1 mentions) Pvdf membrane, by Bio-Rad (1 mentions) Anti β actin, by Merck Group (1 mentions)

9 protocols using pnfκb p65 s536

1

Interrogating Cellular Signaling Pathways in Colorectal Cancer

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Approximately, 30 to 80 mg of nuclear or cytoplasmic protein that was isolated from SW480 colorectal cancer cells using NE-PER nuclear and cytoplasmic extraction kit (Thermo Scientific, Waltham, MA) was loaded and separated by SDS-PAGE, transferred to nitrocellulose membrane, and probed with the following antibodies overnight at indicated dilutions. pNFκB P65 S536, 1:1000, NFκB (P65), 1:1000, pGSK-3αS21βS9, 1:1000, pGSK3αY51βY47, 1:2000, GSK-3β, 1:1000, COX-2, 1:1000, pIKBαS32, 1:1000, IKBα, 1:1000, pIKKα/IKKβS176/180, 1:1000, IKKα/IKKβ, 1:1000, pAktS473, 1:2000, Akt, 1:2000, β-Catenin, 1:1000, pβ-CateninS33/37/T41, 1:1000 (Cell Signaling Technology, Danvers, MA); Each membrane was probed with P84 (Gene Tex, Irvine, CA) to ensure consistent loading of nuclear protein and β-actin, 1:5000 (Sigma, St. Louis, MO) to ensure consistent loading of cytoplasmic protein.
Saud S.M., Li W., Gray Z., Matter M.S., Colburn N.H., Young M.R, & Kim Y.S. (2016). Diallyl Disulfide (DADS), a constituent of garlic, inactivates NFκB and prevents colitis-induced colorectal cancer by inhibiting GSK-3β. Cancer prevention research (Philadelphia, Pa.), 9(7), 607-615.
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2

Western Blot Analysis of Signaling Proteins

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The cells were washed twice with ice-cold PBS and collected with the cell lysis buffer (10 mM Tris-HCl, pH 7.4, 1% SDS, and 1 mM Na3VO4). The cell extracts were sonicated, denatured by heating at 100 °C for 5 min, and quantified with a Dc protein assay kit (Bio-Rad Hercules, CA). Equal aliquots of cell extracts were separated on SDS-polyacrylamide gels. The proteins were then transferred to PVDF membranes (Bio-Rad Hercules, CA), blocked, and probed with one of the antibodies against COX-2 (Cayman Chemical Co.), p-c-Jun S73, p-IKKα/β S176/180, p-IκBα S32/36, p-NFκB p65 S536, total c-Jun IκBα and IKKα/β (Cell Signaling Technology, Beverly, MA), NFAT3, p65, and p50 (Santa Cruz Biotechnology, CA, USA), or β-Actin (Sigma). Primary antibody-bound proteins were detected by using an alkaline phosphatase-linked secondary antibody and an ECF Western blotting system (Amersham, Piscataway, NJ).
Wei J., Du K., Cai Q., Ma L., Jiao Z., Tan J., Xu Z., Li J., Luo W., Chen J., Gao J., Zhang D, & Huang C. (2014). Lead induces COX-2 expression in glial cells in a NFAT-dependent, AP-1/NFκB-independent manner. Toxicology, 325, 67-73.
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3

VCAM-1 and NF-κB Pathway Protein Analysis

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Cell and aorta tissue samples were lysed using RIPA buffer (Cell Signaling Technology, Danvers, MA, USA) and then homogenized using an ultrasonic homogenizer (Hielscher, Germany). The lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred on a polyvinylidene fluoride (PVDF) membrane. Membranes were incubated with specific primary antibodies (1:1000 anti-VCAM-1 or NF-κB p65 or p-NF-κB p65 [s536] and 1:5000 anti-β-actin). Specific antibodies against VCAM-1 (R&D Systems, Minneapolis, MN, USA), nuclear factor-κB (NF-κB) p65, p-NF-κB p65 (s536) (Cell Signaling Technology, Danvers, MA, USA), and anti-β-actin (Sigma-Aldrich, St. Louis, MO, USA) were used in this study.
Kim S., Lee Y.R., Lee E.O., Jin H., Choi Y.H., Joo H.K, & Jeon B.H. (2022). Capsanthin Inhibits Atherosclerotic Plaque Formation and Vascular Inflammation in ApoE−/− Mice. Biomedicines, 10(8), 1780.
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4

Glioma Cell Line Characterization

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FLT, TMZ, and MTT were purchased from Sigma-Aldrich (St Louis, MO, USA). Antibodies to NF-κB/p65 and p-NF-κB/p65 (S536) were purchased from Cell Signaling Technology (Cambridge, MA, USA). MGMT promoter luciferase reporter plasmid pLightSwitch/MGMT was obtained from SwitchGear Genomics Inc (Carlsbad, CA, USA). IκB kinase β (IKKβ) inhibitor (SC-514), monoclonal antibody to MGMT, β-actin, and His-tag were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Human glioma cell lines with high MGMT expression (T98G, U138, SF767, and U251) were obtained from American Type Culture Collection (ATCC, VA, USA) and cultured according to the ATCC protocol. The experimental protocol regarding the use of these human glioma cell lines was approved by the Institutional Research Ethics Committee of Xiangya hospital, Central South University.
Song T., Li H., Tian Z., Xu C., Liu J, & Guo Y. (2015). Disruption of NF-κB signaling by fluoxetine attenuates MGMT expression in glioma cells. OncoTargets and therapy, 8, 2199-2208.
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5

Investigating Fibrotic Signaling Pathways

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Antibodies and reagents were purchased from the following sources: fibronectin (catalog # Ab2413, Abcam, Boston, MA, USA), collagen I (catalog # NB600-408, Novus Biologicals, Centennial, CO, USA), KDEL (catalog # Ab12223, Abcam), ATF4 (catalog # SC-200, Santa Cruz Biotechnology, Dallas, TX, USA), CHOP (catalog # 13172, Novus Biologicals), αSMA (catalog # ab5694, Abcam), pNF-κB p65 (S536) (catalog #3031S, Cell Signaling Technology, Danvers, MA, USA), NF-κB p65 (D14E12) (catalog #8242T, Cell Signaling Technology), MMP9 (N2C1) (catalog #100458, Genetex, Irvine, CA, USA), MMP2(catalog #104577, Genetex), myocilin (catalog # ab41552, Abcam), GAPDH (catalog # 3683, Cell Signaling Technology), and β-actin (catalog # 4970, Cell Signaling Technology). Recombinant human TGFβ2 (catalog # 302-B2-010, R&D systems), astragaloside IV (catalog # 12069, Cayman Chemicals, Ann Arbor, MI, USA). Adenoviral vectors expressing bioactive TGFβ2 or null were purchased from the University of Iowa viral vector core facility (Iowa City, IA, USA).
Kasetti R.B., Maddineni P., Kodati B., Nagarajan B, & Yacoub S. (2021). Astragaloside IV Attenuates Ocular Hypertension in a Mouse Model of TGFβ2 Induced Primary Open Angle Glaucoma. International Journal of Molecular Sciences, 22(22), 12508.
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6

NF-κB Signaling Pathway Analysis

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Tissue and cell extracts were isolated with RIPA buffer (Thermo Fisher, Cat# 89901) supplemented with PhosStop phosphatase inhibitors (Sigma Aldrich, Cat# 4906845001) and cOmpleteTM protease inhibitor mixture (Sigma Aldrich, Cat# 11836153001). Protein extracts were resolved by SDS-PAGE. Blots were incubated overnight at 4°C with antibodies against the following proteins (source, catalog number, and dilution of the antibody are given in parentheses): NF-κB p65 (Cell Signaling Technology, # 6956, 1:500), p-NF-κB p65 (S536) (Cell Signaling Technology, # 3033, 1:500), NLRP3 (Novus, # NBP2-12446, 1:500), Caspase-1 (ABclonal, # A0964, 1:1000), IκBα (Cell Signaling Technology, # 4814, 1:1000), and Lamin B1(ABclonal, # A16909, 1; 500). After washing with TBS-T buffer, blots were incubated with fluorescent (IRDye800 [1:3,000] or IRDye680 [1:3,000], LI-COR Biosciences) conjugated secondary antibodies. The bands were visualized by a Bio-Rad ChemiDoc imaging system (Bio-Rad Laboratories, Inc, USA).
Ren L., Du W., Song D., Lu H., Hamblin M.H., Wang C., Du C., Fan G.C., Becker R.C, & Fan Y. (2022). Genetic ablation of diabetes-associated gene Ccdc92 reduces obesity and insulin resistance in mice. iScience, 26(1), 105769.
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7

Antibody Validation for Protein Analysis

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The following antibodies and reagents were used: Mouse monoclonal and mouse polyclonal MIEN1 (Abnova; antibody specificity tested and proven in previous studies[15 (link), 17 (link)]), rabbit polyclonal MIEN1 (Life Technologies; antibody specificity tested in previous studies[15 (link)]), mouse monoclonal GAPDH (Santa Cruz Biotechnology), rabbit monoclonal pNF-κB p65 S536 and rabbit polyclonal MMP-9 (Cell Signaling Technology), mouse monoclonal VEGF and uPA (R&D Systems), mouse monoclonal Alexa Fluor 594 conjugated Phalloidin (Life Technologies), mouse monoclonal E-cadherin (BD Biosciences), Vimentin (supernatant developed in mouse and tested against human antigen, Developmental Studies Hybridoma Bank), anti-mouse and anti-rabbit IgG (Promega), AlexaFluor 488 goat anti-mouse IgG and AlexaFluor 594 goat anti-mouse IgG (Life Technologies) sheep anti-DIG-AP antibody and NBT-BCIP ready-to-use tablets (Roche), sheep serum (Jackson ImmunoResearch), rabbit IgG, BSA, levamisole hydrochloride, Tris-HCl (pH 7.4), nuclease free water, SSC buffer, Xylene, Tween-20, Nuclear Fast Red, Hematoxylin and Eosin (Sigma-Aldrich) and Permount and PBS (Thermo Fisher Scientific).
Rajendiran S., Parwani A.V., Hare R.J., Dasgupta S., Roby R.K, & Vishwanatha J.K. (2014). MicroRNA-940 suppresses prostate cancer migration and invasion by regulating MIEN1. Molecular Cancer, 13, 250.
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8

LPS-induced CREB and NF-κB signaling

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BMDMs (3 × 106 per well in 6-well cell culture plates) or RAW264.7 cells (2 × 106 per well in 6-well cell culture plates) were treated with LPS 10 or 100 ng/ml for 30 min and lysed in RIPA buffer with protease and phosphatase inhibitors (Roche, Nutley, NJ). Western blot analysis was performed with primary antibodies against p-CREB-S133, CREB, IRS1, IRS2, p-AKT-S473, AKT, p-AKT1-S473, AKT1, p-AKT2-S474, AKT2, p-NF-κB P65-S536, NF-κB P65, IκBα, β-actin, and GAPDH from Cell Signaling Technology (Denvers, MA) and GHSR from Invitrogen (Waltham, MA). Signaling was visualized with ECL (Genesee Scientific, San Diego, CA) and analyzed using Image J software.
Kim D.M., Lee J.H., Pan Q., Han H.W., Shen Z., Eshghjoo S., Wu C.S., Yang W., Noh J.Y., Threadgill D.W., Guo S., Wright G., Alaniz R, & Sun Y. (2023). Nutrient-sensing growth hormone secretagogue receptor in macrophage programming and meta-inflammation. Molecular Metabolism, 79, 101852.
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9

SARS-CoV-2 Infection Signaling Pathway Analysis

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Human cell lines were grown to 70% confluence and lysed in RIPA (10% glycerol, 50mM Tris-HCl pH 7.4, 150mM NaCl, 2mM EDTA, 0.1% SDS, 1% NP40, 0.2% sodium deoxycholate) containing protease and phosphatase inhibitors (Thermo Scientific). Proteins were separated by SDS-PAGE, transferred to a nitrocellulose membrane, blocked in 5% milk, and incubated with primary antibodies overnight at 4°C. Washed membranes were incubated for 45 min at room temperature in secondary antibody solution (LI-COR IRDye 680, 800; 1:10,000 in 5% milk), imaged on an Odyssey® CLx, and analyzed using Image Studio Software. Antibodies were used at the following dilutions: ACE2 (R&D Systems #AF933, 1:200), β-actin (Sigma #A5316, 1:5000), Vinculin (Santa Cruz #sc-73614, 1:2000), AAK1 (Bethyl #A302-146A, 1:1000), AP2M1 (Abcam #ab75995, 1:1000), pAP2M1-T156 (Cell Signaling #3843, 1:1000), SARS-CoV-2-N (Sino Biological #40588-T62, 1:500), pSTAT1-Y701 (Cell Signaling #9167, 1:1000), pSTAT1-S727 (Cell Signaling #8826, 1:1000), STAT1 (Cell Signaling #14994, 1:1000), MX1 (Cell Signaling #37849, 1:1000), IFIT1 (Cell Signaling #14769, 1:1000), pIΚΚα/β-S176/180 (Cell Signaling #2697, 1:1000), IΚΚα (Cell Signaling #11930, 1:1000), IΚΚβ (Cell Signaling #8943, 1:1000), pNFΚB p65-S536 (Cell Signaling #3033, 1:1000), NFΚB p65 (Cell Signaling #8242, 1:1000),
Puray-Chavez M., LaPak K.M., Schrank T.P., Elliott J.L., Bhatt D.P., Agajanian M.J., Jasuja R., Lawson D.Q., Davis K., Rothlauf P.W., Liu Z., Jo H., Lee N., Tenneti K., Eschbach J.E., Shema Mugisha C., Cousins E.M., Cloer E.W., Vuong H.R., VanBlargan L.A., Bailey A.L., Gilchuk P., Crowe JE J.r., Diamond M.S., Hayes D.N., Whelan S.P., Horani A., Brody S.L., Goldfarb D., Major M.B, & Kutluay S.B. (2021). Systematic analysis of SARS-CoV-2 infection of an ACE2-negative human airway cell. Cell Reports, 36(2), 109364.
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