P ikkα

Manufactured by Santa Cruz Biotechnology

Sourced in United States

P-IKKα is a laboratory reagent produced by Santa Cruz Biotechnology. It is a phosphorylated form of the IKKα protein, which plays a role in the NF-kB signaling pathway. The core function of P-IKKα is to serve as a tool for researchers studying the activation and regulation of the NF-kB transcription factor.

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7 protocols using p ikkα

Anti-inflammatory Effects of 6-Shogaol

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6-Shogaol (Sigma-Aldrich, St.Louis, MO, USA), Fetal bovine serum (FBS) (Thermoscientific, USA), RPMI1640 medium (Gibco, Grand Island, NY, USA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT), polyvinylidenedifluoride (PVDF) membranes and enhanced chemiluminescence (ECL) kit (Invitrogen) were used in the study. ELISA kits for determining cytokines-TNF-α, IL-1β and IL-6 were purchased from Biolegend (San Diego, CA, USA). Buffers used in Western blotting analysis were procured from Beyotime Institute of Biotechnology (Beijing, China). Antibodies against VEGF, VEGF-A, VEGFR-2 and COX-2 were procured from Cell Signaling Technology (Danvers, MA, USA). Horseradish peroxidase-labelled IgG secondary antibodies, PGE2, TNF-α, NF-κB p65, IκBα, p-IκBα, p-IKKβ, IKKβ, p-IKKα, IKKα and β-actin were purchased from Santa Cruz Biotechnology (Texas, USA).

Wang D., Jiang Y., Yang X., Wei Q, & Wang H. (2018). 6-Shogaol reduces progression of experimental endometriosis in vivo and in vitro via regulation of VGEF and inhibition of COX-2 and PGE2-mediated inflammatory responses. The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology, 22(6), 627-636.

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Western Blot Analysis of Cellular Proteins

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Cells were lysed with buffer containing 1% NP-40 and proteinase inhibitor cocktail (Thermo Scientific, Rockford, IL). Protein concentrations were determined by Bradford assay (Bio-Rad) and equalized before loading. Cellular protein from each sample was applied to 8% to 12% SDS-PAGE gels and probed with specific antibodies including EEA1, Rab7, RIP1 (Cell Signaling Technology, Danvers, MA), AnxA2, p-IκBα, p-IKKα, p-ERK, p-p38, p-JNK, p-NFκB p65, p-NFκB p50, TNFα, IL-6, IL-1β, and β-actin (Santa Cruz Biotechnology). Blots were developed with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) and chemiluminescent substrate on Fuji X-ray films52 (link).

Zhang S., Yu M., Guo Q., Li R., Li G., Tan S., Li X., Wei Y, & Wu M. (2015). Annexin A2 binds to endosomes and negatively regulates TLR4-triggered inflammatory responses via the TRAM-TRIF pathway. Scientific Reports, 5, 15859.

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Isolation and Characterization of Wogonin

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Wogonin was isolated from S. baicalensis Georgi according to previous protocols35 (link). Wogonin was of ≥99% or higher in all experiments, unless otherwise noted. Wogonin was dissolved in dimethyl sulfoxide (DMSO) as a stock solution (100 mM), stored at −20 °C, and diluted to each of the designated concentrations in the buffer solution before each experiment. The final concentration of DMSO did not exceed 0.1%. ADR were purchased from ZheJiang HiSun Pharmacetuical Co., Ltd. (Zhejiang, China). IM was purchased from Melonepharma (Dalian, China). Primary antibodies of β-actin (1:2000), NF-κB (1:500), p-IKKα (1:500), IKKα (1:500), IκBα (1:500) and p-IκBα (1:500) were obtained from Santa Cruz (Santa Cruz, CA). Nrf2 (1:1000), p-ERK (1:1000), Tubulin (1:1000), Stat3 (1:1000), pY705-Stat3 (1:1000) and Lamin A (1:1000) were from Bioworld (OH, USA). RPMI-1640 (Gibico, Carlsbad, CA) and DAPI (Invitrogen, USA) were purchased. The IRDye^TM 800 conjugated secondary antibodies were the products of Rockland Inc. (Philadelphia, PA). FITC-conjugated anti-human CD13 antibody was purchased from eBioscience. Epidermal growth factor (EGF) was purchased from Sigma, USA.

Xu X., Zhang X., Zhang Y., Yang L., Liu Y., Huang S., Lu L., Kong L., Li Z., Guo Q, & Zhao L. (2017). Wogonin reversed resistant human myelogenous leukemia cells via inhibiting Nrf2 signaling by Stat3/NF-κB inactivation. Scientific Reports, 7, 39950.

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Investigating Anti-inflammatory and Antioxidant Mechanisms

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Antibodies against JAK2, STAT3, p-JAK2, and p-STAT3 were procured from Cell Signaling Technology (Danvers, MA, USA). TNF-α, NF-κB p65, IκBα, p-IκBα, p-IKKα, IKKα, p-IKKβ, IKKβ, horseradish peroxidase-labeled IgG secondary antibodies, and β-actin were purchased from Santa Cruz Biotechnology (Texas, USA). Punicalagin and the buffers used in expression studies were purchased from Sigma-Aldrich (St. Louis, MO, USA). ELISA kits for the determination of cytokines—TNF-α, IL-1β, IL-6, IL-17A, and IL-23—were obtained from BioLegend (San Diego, CA, USA). Levels of ROS production and lipid peroxidation (malondialdehyde content) were measured using kits from Abcam, USA. Superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) enzyme activities were assessed using kits from Abcam, USA. All other reagents and chemicals used in the study were procured from Sigma-Aldrich, otherwise mentioned.

Feng X., Yang Q., Wang C., Tong W, & Xu W. (2020). Punicalagin Exerts Protective Effects against Ankylosing Spondylitis by Regulating NF-κB-TH17/JAK2/STAT3 Signaling and Oxidative Stress. BioMed Research International, 2020, 4918239.

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Western Blot Analysis of Signaling Proteins

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Antibodies were used to detect vimentin (Abcam, Cambridge, MA, and Cell Signaling, Danvers, MA), β-actin (Sigma, St. Louis City, MO), MMP2, MMP7, XIAP, cyclin D1, IL-1β, pSTAT3 (Y705), pSTAT3 (S727), STAT3, NF-κB (p65), p100/52, p105/50, pIkkα, Ikkα/β, pIkBα, IkBα (Santa Cruz Biotechnology), pJAK1, JAK1, pJAK2, JAK2 (Cell Signaling), GAPDH (Merck Millipore, Darmstadt, Germany), and IL-6 (Invitrogen, Carlsbad, CA). Antibodies, manufacturers, catalog numbers, and dilution for specific assays are summarized in Supplemental Table S1.
Cells were lysed with NP-40 lysis buffer supplemented with protease and phosphatase inhibitors (Roche, Manheim, Germany). Total proteins were separated by electrophoresis on a 10% sodium dodecyl sulfate-polyacrylamide gel and transferred to a polyvinylidene fluoride (PVDF) membrane (GE Healthcare, Buckinghamshire, UK) as previously described [11 (link)]. The membrane was probed with each primary antibody at 4°C overnight and with HRP-conjugated secondary antibody (GE Healthcare) for 1 h at room temperature. The signals were detected using enhance chemiluminescence prime Western blotting detection kit (GE Healthcare). Image analysis was performed using Image Quant™ Imager and the band intensity was quantitated with ImageQuant TL software supplied by the manufacturer (GE Healthcare, Uppasala, Sweden). Three independent cultures were used for the analysis.

Saengboonmee C., Phoomak C., Supabphol S., Covington K.R., Hampton O., Wongkham C., Gibbs R.A., Umezawa K., Seubwai W., Gingras M.C, & Wongkham S. (2020). NF-κB and STAT3 co-operation enhances high glucose induced aggressiveness of cholangiocarcinoma cells. Life sciences, 262, 118548.

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Macrophage Inflammatory Mediators Analysis

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RAW 264.7 macrophages were obtained from the Korean Cell Line Bank (KCLB), Seoul, Korea. The growth medium Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and penicillin-streptomycin were obtained from Gibco BRL (Burlington, Canada). Enzyme-linked immunosorbent assay (ELISA) kits for mouse TNF-α, IL-1β, IL-6, and prostaglandin E2 (PGE2) were obtained from R & D system (Minneapolis, MN, USA). TRI Reagent, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Antibodies used in Western blot (iNOS, COX-2, MyD88, IKK-α, p-IKK-α, IκB-α, p-IκB-α, p65, p-p65, p50, p-p50, p38, p-p38, JNK, and p-JNK) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Nagahawatta D.P., Kim H.S., Jee Y.H., Jayawardena T.U., Ahn G., Namgung J., Yeo I.K., Sanjeewa K.K, & Jeon Y.J. (2021). Sargachromenol Isolated from Sargassum horneri Inhibits Particulate Matter-Induced Inflammation in Macrophages through Toll-like Receptor-Mediated Cell Signaling Pathways. Marine Drugs, 20(1), 28.

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Molecular Signaling Pathway Analysis

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Antibodies against PKC-α, PKC-ε and PKC-δ, COX2, p-IKK-α and anti-p-IκB-α were procured from Santa Cruz Biotechnology (Dallas, TX). Antibodies against p-ERK1/2, p-JNK1/2 and p-p38 were purchased from Cell Signaling Technology (Danvers, MA). Antibodies against iNOS and β-actin, lysis buffer and polyvinylidene fluoride (PVDF) membrane were purchased from EMD Millipore (Bedford, MA). LPS (Escherichia coli O111:B4) and protease/phosphatase inhibitor cocktail were purchased from Sigma-Aldrich (St. Louis, MO). Enzyme-linked immunosorbent assay (ELISA) kits and enhanced chemiluminescence (ECL) reagents were purchased from Thermo Fisher Scientific (Waltham, MA). RPMI 1640 was purchased from Gibco Laboratories (Grand Island, NY, USA). Fetal bovine serum (FBS) was purchased from Biological Industries Ltd. (Kibbutz Beit Ha-emek, Israel). L-glutamine was purchased from Life Technologies (Carlsbad, CA, USA). Alamar Blue assay kit was purchased from AbD Serotec (Oxford, UK). QUANTI-Blue medium was purchased from InvivoGen (San Diego, CA).

Ho C.L., Li L.H., Weng Y.C., Hua K.F, & Ju T.C. (2020). Eucalyptus essential oils inhibit the lipopolysaccharide-induced inflammatory response in RAW264.7 macrophages through reducing MAPK and NF-κB pathways. BMC Complementary Medicine and Therapies, 20, 200.

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