Phospho nf κb

Manufactured by Cell Signaling Technology

Sourced in United States, Morocco

Phospho-NF-κB is a primary antibody that detects the phosphorylated form of the NF-κB p65 subunit. NF-κB is a transcription factor that plays a central role in regulating the immune response and inflammatory processes.

Automatically generated - may contain errors

Lab products found in correlation

Close spelling variants of this product

NF-κB (393 citations) Phospho-NF-κB p65 (178 citations) Anti-phospho-NF-κB p65 (Ser536) (40 citations) Rabbit anti-phospho-NF-κB p65 (22 citations) Anti-phospho-NF-κB p65 antibody (11 citations) Anti-phospho-NF-κB antibody (6 citations) Rabbit monoclonal anti-phospho NF-κB p65 (ser536) antibody (5 citations) PathScan® Phospho-NF-κB p65 (Ser536) Sandwich ELISA Kit (4 citations) Anti-phospho-NF-κB p65 (S536) and anti-NF-κB p65 (93H1 and D14E12, (3 citations) Phospho-NF-κB p65 (Ser468) (3 citations)

80 protocols using phospho nf κb

Western Blot Protein Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cell or tissue homogenates were subjected to protein isolation using RIPA lysis buffer, and then protein concentrations were estimated using bicinchoninic acid reagent (BCA-kit from Thermo Scientific, USA). An equal amount of (30 μg) protein from all samples were loaded into SDS-PAGE Bis-Tris 8–10% protein gel for electrophoresis and then transferred onto polyvinylidene difluoride (PVDF) membranes (0.45 μm, Millipore, Billerica, MA, USA). After protein transfer, PVDF membranes were blocked with 5% Bovine serum albumin (Sigma Aldrich, USA) for 1 h at room temperature and then blots were incubated with primary antibodies overnight at 4 °C. Primary antibodies (phospho-NFκB, total NFκB, phospho NFκB, p38, phospho p38, c-JUN) and loading control antibody GAPDH were purchased from Cell signalling technology, Massachusetts, USA. Secondary antibodies (Jackson Laboratory, USA) and an ECL kit (Advansta, Menlo park, CA, USA) were employed to generate chemiluminescent signals. ReBlot Plus Strong Antibody Stripping Solution (Millipore, USA) was used to re-probe the blots. All immunoblot quantifications were representing the triplicate repeats. Densitometry analysis was performed using ImageJ software.

Tirunavalli S.K., Gourishetti K., Kotipalli R.S., Kuncha M., Kathirvel M., Kaur R., Jerald M.K., Sistla R, & Andugulapati S.B. (2021). Dehydrozingerone ameliorates Lipopolysaccharide induced acute respiratory distress syndrome by inhibiting cytokine storm, oxidative stress via modulating the MAPK/NF-κB pathway. Phytomedicine, 92, 153729.

+ Open protocol

+ Expand

EGCG Modulation of Inflammatory Pathways

Check if the same lab product or an alternative is used in the 5 most similar protocols

EGCG (purity > 95%) was obtained from Huzhou Rongkai Foliage Extract Co., Ltd. (Huzhou, China). A fatty acid-albumin complex solution containing palmitic acid (PA, Sigma–Aldrich, St Louis, MO, USA) and fatty acid-free bovine serum albumin (BSA, Sigma, St Louis, MO, USA) was prepared as described previously [44 (link)]. All cell culture reagents were purchased from Gibco (Grand Island, NY, USA). Primary antibodies specific for NF-κB, phospho-NF-κB, IκB-α, phospho-IκB-α, Stat3, phosphoStat3, Jak2, and phospho-Jak2 were purchased from Cell Signaling Technology (Beverly, MA, USA), and Iba-1 was obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). D12450J normal chow diet and D12492 high-fat diet were purchased from Research Diets, Inc. Co., Ltd. (New Brunswick, NJ, USA).

Mao L., Hochstetter D., Yao L., Zhao Y., Zhou J., Wang Y, & Xu P. (2019). Green Tea Polyphenol (−)-Epigallocatechin Gallate (EGCG) Attenuates Neuroinflammation in Palmitic Acid-Stimulated BV-2 Microglia and High-Fat Diet-Induced Obese Mice. International Journal of Molecular Sciences, 20(20), 5081.

+ Open protocol

+ Expand

Immunoblotting and Immunofluorescence Antibody Protocol

Check if the same lab product or an alternative is used in the 5 most similar protocols

Immunoblot analyses were performed using the following antibodies: Akt, Aktp^S473, p70S6K, phospho-p70S6K, 4EBP1, ERK1/2, Atg5, ULK, ERK1/2, phopho-ERK1/2, p38, phospho-p38, phospho-NFκB and phospho-4EBP1 were all purchased from Cell Signaling. FAK (Santa Cruz Biotechnology, Inc.), actin (Cytoskeleton), LC3 (Novus) and FAKp^Y397 (BD Transduction Laboratories) were purchased from the suppliers indicated. Monoclonal anti-HA (16B12) and polyclonal anti-HA (HA.11) were both purchased from Covance. For immunofluorescence studies, LC3 (Millipore), mTOR (Cell Signaling), LAMP-1 and LAMP-2 (Developmental Studies Hybridoma Bank, University of Iowa), p62 (abcam) and ubiquitin (FK2, Enzo Life Sciences) were from the suppliers indicated. Secondary antibodies included goat anti-mouse-Cy3, donkey anti-rabbit Alexa Fluor 488 and goat anti-rat Alexa Fluor 555 and were purchased from Invitrogen. For flow cytometry, TLR4 (Sa15-21; Akashi et al., 2003) was conjugated to biotin and was a kind gift from Jonathan Kagan (Harvard Medical School, Boston, MA). Anti- strepavidin-APC was purchased from Biolegend.

Owen K.A., Meyer C.B., Bouton A.H, & Casanova J.E. (2014). Activation of Focal Adhesion Kinase by Salmonella Suppresses Autophagy via an Akt/mTOR Signaling Pathway and Promotes Bacterial Survival in Macrophages. PLoS Pathogens, 10(6), e1004159.

+ Open protocol

+ Expand

Whole Cellular Protein Extraction and Immunoblotting

Check if the same lab product or an alternative is used in the 5 most similar protocols

For extraction of whole cellular proteins, cells were washed twice with ice-cold phosphate buffered saline and then lysed with cell lysis buffer (50 mM Tris-HCl pH 7.4, 1% NP-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA, 10 mM Na3VO4, 50 mM NaF, 1 mM PMSF, 1 µg/mL aprotinin, 1 µg/mL leupeptin, 1 µg/mL pepstatin) on ice for 30 minutes. Lysates were sonicated, and the cell homogenates were centrifuged at 15,000 g for 10 minutes (4℃). After centrifugation, supernatants were electrophoresed in 10% acrylamide gels and transferred onto nitrocellulose membranes. The proteins were probed overnight with antibodies against JNK, ERK p44/42, p38, NF-κB p65, phospho-JNK, phospho-ERK p44/42, phospho-p38, phospho-NF-κB (Cell Signaling Technology, Danvers, MA, USA) and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The immunoreactive bands were detected with horseradish peroxidase-conjugated secondary antibodies and visualized by enhanced chemiluminescence.

Song Y., Hong S., Iizuka Y., Kim C.Y, & Seong G.J. (2015). The Neuroprotective Effect of Maltol against Oxidative Stress on Rat Retinal Neuronal Cells. Korean Journal of Ophthalmology : KJO, 29(1), 58-65.

+ Open protocol

+ Expand

Pulmonary Endothelial Cell Culture Protocol

Check if the same lab product or an alternative is used in the 5 most similar protocols

Human pulmonary artery endothelial cells (HPAECs) were obtained from Lonza (Allendale, NJ) and used at passages 5–8. All experiments were performed in EGM growth medium (Lonza) containing 2% fetal bovine serum (FBS) unless otherwise specified. Texas Red–conjugated phalloidin and Alexa Fluor 488–labeled secondary antibodies were purchased form Molecular Probes (Eugene, OR). TRAP6 was obtained from AnaSpec (San Jose, CA). 8CPT was obtained from Calbiochem (La Jolla, CA). GGTI-298 and thrombin were obtained from Millipore-Sigma. Antibodies to diphospho–myosin light chain (MLC), pan-MLC, MYPT, pMYPT, p120-catenin, phospho-NFκB, β-actin, and tubulin antibodies were obtained from Cell Signaling (Beverly, MA); Rap1, VE-cadherin, ICAM1, VCAM1, and E-selectin antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). HRP-linked anti-mouse and anti-rabbit IgG were obtained from Cell Signaling (Beverly, MA). Unless otherwise specified, all biochemical reagents were obtained from Sigma (St. Louis, MO).

Ke Y., Karki P., Zhang C., Li Y., Nguyen T., Birukov K.G, & Birukova A.A. (2019). Mechanosensitive Rap1 activation promotes barrier function of lung vascular endothelium under cyclic stretch. Molecular Biology of the Cell, 30(8), 959-974.

+ Open protocol

+ Expand

Western Blot Analysis of Signaling Pathways

Check if the same lab product or an alternative is used in the 5 most similar protocols

Harvested cells were lysed in TBSN buffer (20 mM Tris, pH 8.0, 150 mM NaCl, 1.5 mM EDTA, 5 mM EGTA, 0.5% Nonidet P-40, and 0.5 mM Na₃VO₄) supplemented with proteinase inhibitors. The lysates were resolved by SDS-PAGE and transferred to Whatman Westran PVDF membrane (Sigma, Z671088), followed by incubation with antibodies against Plk1 (Santa Cruz, sc-17783), phospho-AKT-S473 (Cell Signaling, 9271), phospho-AKT-T308 (Cell Signaling, 13038), AKT (Cell Signaling, 9272), phospho-S6K (Cell Signaling, 9205), phospho-S6 (Cell Signaling, 2211), S6 (Cell Signaling, 2217), phospho-NFκB (Cell Signaling, 3033), NFκB (Santa Cruz, sc-372), AR (Santa Cruz, sc-7305), γ-tubulin (Sigma, T3559), p84 (Abcam, ab487), Twist1 (Sigma, SAB1411370), SREBP1 (Santa Cruz, sc-367), SREBP2 (Santa Cruz, sc-5603), phospho-GSK3β (Cell Signaling, 9322), GSK3β (BD Biosciences, 610201), FAS (BD Biosciences, 610962), HMG-CoA Reductase (EMD Millipore, ABS229), cleaved-PARP (EMD Millipore, AB3620), PSA (Cell Signaling, 5365), and β-actin (Sigma, A5441).

Zhang Z., Hou X., Shao C., Li J., Cheng J.X., Kuang S., Ahmad N., Ratliff T, & Liu X. (2014). Plk1 inhibition enhances the efficacy of androgen signaling blockade in castration-resistant prostate cancer. Cancer research, 74(22), 6635-6647.

+ Open protocol

+ Expand

Protein Extraction and Western Blotting of 4T1 Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

Total protein of murine 4T1 cells was extracted using RIPA lysis buffer (50 mM Tris, 150 mM sodium chloride, 1% NP-40, 0.25% deoxycholate) supplemented with protease and a phosphatase inhibitor cocktail (Beyotime Biotechnology). Equal amounts of protein were separated by 8–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Merck Millipore, Burlington, MA, USA) by electroblotting. The membranes were probed with primary antibodies against B cell lymphoma 2-associated X (BAX; 1:1000, Zen-bioscience, Chengdu, Sichuan, China), IκBα, phospho-IκBα, NF-κB, phospho-NF-κB (1:1000 Cell Signaling Technology). Blots were developed with HRP-conjugated secondary antibodies and chemiluminescent substrates on Kodak X-ray film. The density of electrophoresis bands was quantified for statistical analysis.

Liu Y., Wang L., Liu J., Xie X., Hu H, & Luo F. (2020). Anticancer Effects of ACT001 via NF-κB Suppression in Murine Triple-Negative Breast Cancer Cell Line 4T1. Cancer Management and Research, 12, 5131-5139.

+ Open protocol

+ Expand

Investigating Diabetic Inflammation with STZ and Vitamin D

Check if the same lab product or an alternative is used in the 5 most similar protocols

Streptozotocin (STZ) and 1,25(OH)₂D₃ were purchased from Sigma–Aldrich (St. Louis, MO, USA). Polyclonal antibodies targeting rat TLR4, TNF-α, NF-κB p65, phospho-NF-κB, and β-actin were purchased from Cell Signaling Technology (Danvers, MA, USA). Protamine zinc insulin (P) was obtained from Jiangsu Wanbang Biological Technology Ltd. (Xuzhou, Jiangsu, China). CD68 was purchased from Wuhan Boster Biological Technology Ltd. (Wuhan, Hubei, China).

Wang H., Zhang Q., Chai Y., Liu Y., Li F., Wang B., Zhu C., Cui J., Qu H, & Zhu M. (2015). 1,25(OH)2D3 downregulates the Toll-like receptor 4-mediated inflammatory pathway and ameliorates liver injury in diabetic rats. Journal of Endocrinological Investigation, 38, 1083-1091.

+ Open protocol

+ Expand

Western Blot Analysis of Inflammatory Signaling

Check if the same lab product or an alternative is used in the 5 most similar protocols

After appropriate treatments and rinsing with cold phosphate-buffered saline, REC were collected in lysis buffer containing protease/phosphatase inhibitors and scraped into their respective tubes. Retinal extracts were prepared by sonication. Equal amounts of protein from cell or tissue extracts were separated on pre-cast tris-glycine gels (Invitrogen, Carlsbad, CA) and blotted onto a nitrocellulose membrane. After blocking in TBST (10mM Tris-HCl buffer, pH 8.0, 150 mM NaCl, 0.1% Tween 20) and 5% (w/v) BSA, membranes were treated with the following primary antibodies: TLR4, IL-1 receptor-associated kinase 1 (IRAK1), TNF receptor-associated factor 6 (TRAF6), MyD88, HMGB1 (Abcam, San Francisco, CA), NF-κB, phospho-NF-κB (Cell Signaling, Danvers, MA), and β-actin (Santa Cruz, Santa Cruz, CA); then followed by incubation with secondary antibodies (Fisher Scientific, Pittsburgh, PA) labeled with horseradish peroxidase. Antigen-antibody complexes were detected using a chemilluminescence reagent kit (Thermo Scientific, Pittsburgh, PA). Western blot images were collected on an Azure Biosystem C500 machine (Azure Biosystems, Dublin, CA). Mean densitometry of bands for each protein were measured.

Berger E.A., Carion T.W., Jiang Y., Liu L., Chahine A., Walker R.J, & Steinle J.J. (2016). β-adrenergic receptor agonist, Compound 49b, inhibits TLR4 signaling pathway in diabetic retina. Immunology and cell biology, 94(7), 656-661.

+ Open protocol

+ Expand

Molecular Signaling Pathway Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

Diethylnitrosamine (DEN) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Antibodies against IRS1, phospho-IRS-1(Ser1101), NF-κB, phospho-NF-κB, phospho-AKT(Ser473), phospho–AKT (Thr308), JNK, phospho-SAPK/JNK (Thr183/Tyr185), phospho-p38MARK (Thr180/Tyr182), phospho-p44/42 MARK (Erk1/2) (Thr202/Tyr204), mTOR, phospho-mTOR, and GAPDH were purchased from Cell Signaling Technology (USA). The bicinchoninic acid (BCA) protein assay kit and the phosphatase inhibitor tablets were purchased from Thermo Fisher Scientific (USA).

Zhou Y., Liu Z., Lynch E.C., He L., Cheng H., Liu L., Li Z., Li J., Lawless L., Zhang K.K, & Xie L. (2020). Osr1 regulates hepatic inflammation and cell survival in the progression of non-alcoholic fatty liver disease. Laboratory investigation; a journal of technical methods and pathology, 101(4), 477-489.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!