Pnfkb

Manufactured by Cell Signaling Technology

Sourced in United States

PNFkB is a laboratory equipment product manufactured by Cell Signaling Technology. It is designed to detect and quantify the levels of phosphorylated NF-κB, a transcription factor that plays a central role in regulating immune and inflammatory responses. The product provides a reliable and sensitive tool for researchers to study the activation and signaling pathways of NF-κB in various biological systems.

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Lab products found in correlation

29 protocols using pnfkb

Comprehensive Western Blot Analysis

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Cells were lysed in RIPA buffer (Beyotime, Jiangsu, China) containing a protease inhibitor cocktail (Sigma, St. Louis, CA, USA). Total protein was assessed using a BCA Protein Assay Kit (Beyotime). Western blot analysis was performed as previously described [18 (link)]. Antibodies against the following were used in this study, PABPC1 (1:1000, Abcam, SF, USA), IFI27, HA tag, Flag tag, TSG101, CD63, CD9, PCNA, Alix, Ki67, caspase 3, CXCL10, CD34, cleaved-PARP, PARP, eIF4G, cleaved caspase 9, caspase 9, ERK, p-ERK, IFI27, STAT3, p-STAT3, STAT1, p-STAT1, NF-kB, p-NF-kB, β-actin and GAPDH (all 1:1000, Cell Signaling Technology, MA, USA), EXOSC2 and EXOSC4 (both 1:1000, Santa Cruz, MA, USA).

Zhang Y., Chen C., Liu Z., Guo H., Lu W., Hu W, & Lin Z. (2022). PABPC1-induced stabilization of IFI27 mRNA promotes angiogenesis and malignant progression in esophageal squamous cell carcinoma through exosomal miRNA-21-5p. Journal of Experimental & Clinical Cancer Research : CR, 41, 111.

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Reverse Western Blot Analysis of Angiogenic Factors

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For reverse western blot, Proteome Profiler Antibody Array Kits for human angiogenesis factors, chemokines, cytokines and phospho-kinases (R&D systems) were used, according to the manufacturer’s instructions. For western blot, 400,000 MDA-MB-231 or LECs (per well) were starved for 24 h, after which they were treated with Stattic (5–10 µM), S3I-201 (2.5–10 µM), or SP600125 (40 µM) and incubated for 60 min. After that, inducers, including TCM (30%), EGM, IL6-dep-TCM, IL6 or EGF were added. We followed the standard protocol for the rest of the procedure as described previously^{11 (link)} applying antibodies of interest, including p Stat3, HIF-1α, gp130, p NFkB, NFkB, IkBα, Stat3, pCREB, GAPDH (all from Cell Signaling), pc-Jun, pATF-2 (Sigma), CCR5, and Lamin B1 (Abcam). All the original gel images of immunoblot analyses are presented in Supplementary Fig. 25.

Lee E., Fertig E.J., Jin K., Sukumar S., Pandey N.B, & Popel A.S. (2014). Breast cancer cells condition lymphatic endothelial cells within pre-metastatic niches to promote metastasis. Nature communications, 5, 4715.

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Characterization of AITR-Mediated Intracellular Signaling

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EXAMPLE 9

Purified CD4⁺ T cells (1×10⁶cells/well) were stimulated with anti-CD3 antibody and IL-2 for 3 days. The cultured cells were treated with anti-AITR mAbs for 2, 6, 12 and 24 hours. Then the cells were harvested and the suspension thereof was prepared. Levels of phospho-STATs (STAT1, STAT2, STAT3, STAT4, STAT5, and STAT6) and master transcription factors (T-bet, GATA-3, RORγt and Foxp3) were analyzed by flow cytometry. The cells in the culture were lysed with RIPA buffer. Subsequently, total protein extract was resolved on 8% to 12% SDS-polyacrylamide gel electrophoresis and immunoblotted with antibodies against NFAT1/2, p-p38, p-ERK1/2, p-JNK1/2, and p-NF-KB (Cell Signaling, Danvers, Mass., USA). The same blot was re-probed with an anti-β-actin antibody as a control for protein loading.

In a separate experiment, the sorted CD4⁺CD25^highT cells (5×10⁵cells/well) were stimulated with 5 μg/ml anti-AITR mAbs and phosphorylation of the proteins involved in intracellular signaling pathway was analyzed by the same method described above.

US11053298B2. Epitope for switching to T_H2 cell and use thereof (2021-07-06). Eutilex Co., Ltd. [KR]. Inventors: Byoung S. Kwon [KR].

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Immunoblot Analysis of Signaling Pathways

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Lipopolysaccharides from Escherichia coli O55:B5 (LPS), horseradish peroxidase (HRP) conjugated anti-rabbit, and anti-mouse antibodies, immunoblot chemicals, and protease inhibitor cocktail were purchased from Sigma Chemicals (MO, USA). Primary antibodies for NF-kB, pNF-kB, PI3K, AKT, pAKT, ATG5, LC3, BAX, caspase 3, and caspase 12 were purchased from Cell Signaling Technology, (Massachusetts, USA). TNFa, pERK1, pJNK1, pPI3K, GRP78, pIRE1, CHOP, Beclin1, P62, Bcl2, and β-actin antibodies were purchased from Thermo Scientific (Massachusetts, USA).

Nagaraju M., Kalahasti K.K., Reddy K.P., Addi U.R., Satyavani M., Reddy G.B, & Reddy S.S. (2022). Anti-inflammatory potential of turmeric, amla, and black pepper mixture against sepsis-induced acute lung injury in rats. Journal of Food Science and Technology, 60(1), 252-261.

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Antibody Characterization and NFkB Inhibition

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The Twist2 antibody was purchased from Abcam (#ab66031) and used at 1:5000 for immunoblotting, 1:1000 for immunohistochemistry and 1:500 for immunofluorescence. Antibodies for NFkB (1:1000; #4764), pNFkB (1:1000; #3033), ITGA1 (2 μg/ml; #71,747), Vimentin (1:5000; #9855), and E-cadherin (1:1000; #3195) were purchased from Cell Signaling Technology. The LAMA4 (1:1000; ab205568) and Fibronectin (1:15,000; ab45688) antibodies were purchased from Abcam. The RhoC antibody, used at a 1:1000 dilution, was graciously shared by Dr. Kenneth van Golen (University of Delaware). The β-actin antibody (1:10,000; #A5316) was used as a loading control for immunoblotting and was purchased from Sigma-Aldrich Corp. AlexaFluor488 anti-rabbit or AlexaFluor 568 anti-mouse secondary antibodies were used for Western blots scanned with the Typhoon FLA 9000. An AlexaFluor488 anti-rabbit secondary antibody was used for immunofluorescence. Inhibition of NFkB was performed using NFkB Activation Inhibitor II, JSH-23 (#481,408; Millipore) at a concentration of 10 μM for 2 h.

Lehman H.L., Kidacki M, & Stairs D.B. (2020). Twist2 is NFkB-responsive when p120-catenin is inactivated and EGFR is overexpressed in esophageal keratinocytes. Scientific Reports, 10, 18829.

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Antibody-Based Protein Expression Assay

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Antibodies for STAT3, phospho-STAT3, p-NFKB, cyclin D1, cleaved caspase-3 (CC3), c-Myc, Ki67, GAPDH, Ubiquitin and horseradish peroxidase-conjugated secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA). The CellTiter 96® AQ_ueous One Solution (MTS) Reagent containing 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium was obtained from Promega (Madison, WI).

Njatcha C., Farooqui M., Kornberg A., Johnson D.E., Grandis J.R, & Siegfried J.M. (2018). STAT3 Cyclic Decoy Demonstrates Robust Antitumor Effects in Non-Small Cell Lung Cancer. Molecular cancer therapeutics, 17(9), 1917-1926.

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Investigating Transcription Factor Activation

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LECs (2×10⁶) treated with Stattic, SP600125, IL6 or EGM, were used to prepare cell lysates or nuclear extracts. Five hundred microliters of cell lysates or 200 µL nuclear extracts were incubated overnight at 4°C with antibodies suitable for IP (1:100 diluted): pStat3, pc-Jun, pATF-2, pNFkB, and NFkB (Cell Signaling). Ten microliters of Protein A/G Plus Agarose (Santa Cruz Biotech) was added and incubated for 3 h at 4°C. The beads were rinsed 3 times with 500 µL cell lysis buffer for IP (Pierce) supplemented with the protease inhibitor and phosphatase inhibitor cocktail 2/3 (Sigma). The protein complex was reduced and separated by SDS-PAGE and probed with the following antibodies in a Western assay: pStat3, pc-Jun, pATF-2, pNFkB, and NFkB (Sigma). All the original gel images of immunoblot analyses are presented in Supplementary Fig. 25.

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Western Blotting of Cell Signaling Proteins

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Western blotting was performed as previously described (17 (link)). The following specific antibodies were used: XIAP (1:1000; cat. no. 2042; Cell Signaling Technology, Danvers, MA, USA), cIAP2 (1:1000; cat. no. 3130; Cell Signaling Technology), p-NF-kB (1:1000; cat. no. 3033; Cell Signaling Technology), NF-kB (1:1000; cat. no. 8242; Cell Signaling Technology), cIAP1 (1:1000; cat. no. sc-271419; Santa Cruz Biotechnology, Dallas, TX, USA), and β-actin (1:1000; cat. no. sc-47778; Santa Cruz Biotechnology).

Prasopporn S., Suppramote O., Ponvilawan B., Jamyuang C., Chanthercrob J., Chaiboonchoe A., More-Krong P., Kongsri K., Suntiparpluacha M., Chanwat R., Korphaisarn K., Okada S., Sampattavanich S, & Jirawatnotai S. (2022). Combining the SMAC mimetic LCL161 with Gemcitabine plus Cisplatin therapy inhibits and prevents the emergence of multidrug resistance in cholangiocarcinoma. Frontiers in Oncology, 12, 1021632.

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Western Blot Analysis of Conjunctival pNF-kB

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The animals were euthanized using CO₂ asphyxiation and the mouse conjunctival tissues were harvested. The conjunctiva sample underwent cold PBS washing followed by incubation in lysis buffer containing phosphatase and protease inhibitors. Tissue lysates were sonicated and homogenized, then centrifuged to obtain supernatants for protein quantification. Electrophoresis on SDS gels, protein transfer to PVDF membranes, and blocking with BSA in TBST buffer were conducted. After that, membranes were incubated overnight at 4 °C with specific rabbit polyclonal antibodies against phosphorylated NF-kB (pNF-kB; 1:1000, Catalog No. 3033S; Cell Signaling Technology, Inc.) and GAPDH (1:10,000, Catalog No. 2118S; Cell Signaling Technology, Inc.). The membranes were washed and incubated with an HRP-linked secondary antibody, and the bands were visualized with a chemiluminescence system (WBKLS0100; MilliporeSigma, Inc., Burlington, MA, USA). The protein expression levels were normalized to GAPDH in the same samples.

Bhujel B., Oh S., Hur W., Lee S., Chung H.S., Lee H., Park J.H, & Kim J.Y. (2024). Effect of Exposure to Particulate Matter on the Ocular Surface in an Experimental Allergic Eye Disease Mouse Model. Bioengineering, 11(5), 498.

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Immunohistochemical and Immunocytochemical Analyses

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Paraffin-embedded slides were deparaffinized, dehydrated, and rehydrated using a series of acetone and alcohol washes. Antigen retrieval was performed using citrate buffer at pH 6.0 in a pressure cooker. The primary antibody (PTGS2: 160112, 1:100; Cayman Chemicals) was prepared in 4% fish gelatin and incubated with the slides overnight at 4°C. Slides were then washed and incubated with HRP-conjugated secondary antibody (Jackson Immuno Research, West Grove, PA) at room temperature for 1 hour. DAB substrate (Invitrogen, Grand Island, NY) was used to visualize staining of protein and hematoxylin (Invitrogen) was used to stain nuclei. For immunocytochemistry experiments, Skov3-ip1 and HeyA8 cells were grown in chamber slides prior to treatment with NE. Cells were fixed in 4% paraformaldehyde and permeabilized using Triton X-100. Staining using primary (PTGES: 1:100; T6079, Sigma Aldrich, p-Nf-kB: 1:100; 3033S, Cell Signaling, total Nf-kB: 1:100, 8242S, Cell Signaling) and secondary antibodies (anti-rabbit Alexa Flour 594, 1:800; Jackson Immuno Research) was done as described above. Hoechst (Invitrogen) was used to stain nuclei.

Nagaraja A.S., Dorniak P.L., Sadaoui N.C., Kang Y., Lin T., Armaiz-Pena G., Wu S.Y., Rupaimoole R., Allen J.K., Gharpure K.M., Pradeep S., Zand B., Previs R.A., Hansen J.M., Ivan C., Rodriguez-Aguayo C., Yang P., Lopez-Berestein G., Lutgendorf S.K., Cole S.W, & Sood A.K. (2015). Sustained adrenergic signaling leads to increased metastasis in ovarian cancer via increased PGE2 synthesis. Oncogene, 35(18), 2390-2397.

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