Ikkα β

Manufactured by Santa Cruz Biotechnology

Sourced in United States

IKKα/β is a laboratory product offered by Santa Cruz Biotechnology. It functions as a key component in the IKK complex, which is responsible for the phosphorylation and subsequent degradation of IκB proteins. This process regulates the activity of the transcription factor NF-κB, a central mediator of inflammatory and immune responses.

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P-IKKα/β (9 citations) Anti-IKKα/β (8 citations) IKKα/β (sc-7607, (6 citations)

16 protocols using ikkα β

Fractalkine Signaling Pathway Assay

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Anti-mouse and anti-rabbit IgG-conjugated horseradish peroxidase and rabbit polyclonal antibodies specific for fractalkine/CX3CL1, CX3CR1, ICAM-1, VCAM-1, p-PI3K p85α, PI3K p85α, p-Akt, Akt, p-IKKα/β, IKKα/β, p-IκBα, IκBα, p-p65, p65, and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Recombinant human fractalkine/CX3CL1 was purchased from PeproTech (Rocky Hill, NJ, USA). The short hairpin RNA (shRNA) plasmid used for gene knockdown was purchased from the National RNAi Core Facility Platform (Taipei, Taiwan). All siRNAs used were ON-TARGETplus siRNAs and purchased from Dharmacon Research (Lafayette, CO, USA). All other chemicals were obtained from Sigma–Aldrich (St. Louis, MO, USA).

Liu J.F., Tsao Y.T, & Hou C.H. (2016). Fractalkine/CX3CL1 induced intercellular adhesion molecule-1-dependent tumor metastasis through the CX3CR1/PI3K/Akt/NF-κB pathway in human osteosarcoma. Oncotarget, 8(33), 54136-54148.

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Western Blot Analysis of Inflammatory Markers

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Protein from microglia or ischemic penumbra was centrifuged at 13,000 × g at 4°C for 30 min and quantified through a BCA kit (P0011, Beyotime Biotech). Equal amounts of protein were loaded onto 10% SDS‐PAGE gel (PG212, Epizyme), separated by electrophoresis and transferred to Polyvinylidene fluoride membranes (PVDF, 1620177, Bio‐Rad). The membrane was blocked by 5% non‐fat milk for 2 h and incubated with primary antibodies against IL‐1β (sc52012, Santa Cruz), IL‐6 (sc‐28343, Santa Cruz), TNF‐α (sc‐52746, Santa Cruz), iNOS (BS40374, Bioworld Biotechnology), p‐NF‐κB/p‐P65 (3033, Cell Signaling Technology), NF‐κB/P65 (8242, Cell Signaling Technology), IKKα/β (sc‐8032, Santa Cruz), p‐IKKα/β (2697, Cell Signaling Technology), IκBα (BS3601, Bioworld Biotechnology), p‐IκBα (2859, Cell Signaling Technology), NEMO (ab178872, abcam), anti‐Ubiquitin (ab134953, abcam), anti‐SUMO (ab32058, abcam), COX‐2 (BS1076, Bioworld Biotechnology) or β‐actin (BS40736, Bioworld Biotechnology) overnight at 4°C. On the following day, the membrane was immersed in the corresponding secondary antibodies (BS22357/BS22356, Bioworld Biotechnology) on the shaker for 1–2 h. After moistened with the enhanced chemiluminescence (ECL, 34580, Thermo Fisher Scientific), the protein band was visualized through Gel‐Pro system (Tanon Technologies) and analyzed using ImageJ (ImageJ 1.5, NIH).

Lan Z., Qu L., Liang Y., Chen L., Xu S., Ge J., Xue Z., Bao X., Xia S., Yang H., Huang J., Xu Y, & Zhu X. (2024). AZD1390, an ataxia telangiectasia mutated inhibitor, attenuates microglia‐mediated neuroinflammation and ischemic brain injury. CNS Neuroscience & Therapeutics, 30(4), e14696.

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Liver Protein Quantification via Western Blot

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Liver proteins were quantified by western blot analysis as follows using BioRad, (Hercules, CA) Criterion and ChemiDoc systems following manufacturers’ instructions. PGC1α antibody was purchased from EMD Millipore, Billerica, MA, Cat# ST1202. All other antibodies purchased from Santa Cruz Biotechnology, Santa Cruz, CA were as follows: FOXO1Ser256 Cat# sc-101681; G6Pase-α Cat# sc-27198; PEPCK Cat# sc-32879; PPARα Cat# sc-9000; SREBP-1 Cat# sc-13551; mTORC Cat# sc-8319; PPARγ Cat# sc-7273; PGC1β Cat# sc-67286, NFκB p65 Cat# sc-8008; IKKαβ Cat# sc-7607; SOCS3 Cat# sc-9023; JNK Cat# sc-571; Actin Cat# sc-47778. Criterion gradient tris-glycine precast gels, secondary antibodies, PVDF blotting membranes, molecular weight markers, and Enhanced Chemiluminescence (ECL) reagents were also purchased from BioRad. Samples from 8 SHR Vehicle, 8 SHR Bromocriptine treated rats, and 6 Wistar wild type controls were loaded onto the same 26 well Criterion gel along with the molecular weight markers; band intensity was compared only within the samples loaded onto the same gel. A housekeeping protein (Actin) was concurrently quantified on all gels, and the test protein amount was normalized to Actin in the Western blot analysis. Protein bands were quantified with BioRad ImageLab 4.1 software.

Ezrokhi M., Luo S., Trubitsyna Y, & Cincotta A.H. (2014). Neuroendocrine and metabolic components of dopamine agonist amelioration of metabolic syndrome in SHR rats. Diabetology & Metabolic Syndrome, 6, 104.

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Inflammatory Signaling Pathway Modulation

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3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), and all other chemicals were purchased from Millipore Sigma (Billerica, MA, USA). Recombinant human TNF-α and recombinant human IFN-γ were purchased from Bio-Techne Ltd. (Abingdon, UK). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin, and streptomycin were obtained from Life Technologies Inc. (Grand Island, NY, USA). Primary antibodies against p-IKK α/β (cat no. 2697), NF-κB p65 (cat no. 8242), p-Akt (cat no. 9271), and ICAM-1 (cat no. 4915) were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). Primary antibodies against IKK α/β (cat no. 7607), p-IκB-α (cat no. 8404), IκB-α (cat no. 203), Akt1/2/3 (cat no. sc-8312), PARP (cat no. sc-9542), α-tubulin (cat no. sc-8035), Filaggrin (cat no. sc-66192), VCAM-1 (cat no. sc-1504), E-selectin (cat no. sc-5262), and β-actin (cat no. sc-81178) were purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). Horseradish peroxidase-conjugated secondary antibodies were purchased from Jackson ImmunoResearch laboratories, Inc. (West Grove, PA, USA). The histamine ELISA kit was obtained from Enzo life Sciences, Inc. (Farmingdale, NY, USA). The ELISA kits for TNF-α and IL-6 were obtained from R&D Systems, Inc. (Minneapolis, MN, USA).

Kang Y.M., Lee K.Y, & An H.J. (2018). Inhibitory Effects of Helianthus tuberosus Ethanol Extract on Dermatophagoides farina body-induced Atopic Dermatitis Mouse Model and Human Keratinocytes. Nutrients, 10(11), 1657.

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Puerarin Modulates Inflammatory and Apoptotic Pathways

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Puerarin-V (HPLC, 98%) was provided as a lyophilized powder by the Institute of Materia Medica (Beijing, China). Puerarin injection was purchased from Beijing Shkb Pharmaceutical Co., Ltd. (Beijing, China). ISO and LPS were purchased from Sigma-Aldrich (St Louis, MO, USA). AST and LDH kits were purchased from BioSino Biotechnology & Science Inc (Beijing, China). TRIzol^® for total RNA extraction was purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). PrimeScriptTM RT Reagent Ki and SYBR^® Premix Ex TaqTM were purchased from TaKaRa (Shiga, Japan). The antibodies against P-NF-κBp65, NF-κBp65, p-IκB-α, IκB-α, and GAPDH were purchased from Cell Signaling Technology (Danvers, MA, USA). The antibodies against PPAR-γ, p-IKKα/β, and IKKα/β were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The antibodies against Bax, Bcl-2, and caspase 3 were purchased from Abcam (Cambridge, MA, USA). The anti-rabbit IgG or anti-mouse IgG secondary antibodies, enhanced chemiluminescence (ECL) and loading buffer were purchased from CWBIO (Beijing, China). All other chemicals used in this study are commercially available.

Li X., Yuan T., Chen D., Chen Y., Sun S., Wang D., Fang L., Lu Y, & Du G. (2018). Cardioprotective Effects of Puerarin-V on Isoproterenol-Induced Myocardial Infarction Mice Is Associated with Regulation of PPAR-Υ/NF-κB Pathway. Molecules, 23(12), 3322.

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Investigating Intracellular Signaling Pathways

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LPS, D-galactosamine, PD98059 and U0126 were obtained from Sigma (St. Louis, MO). Anti-MEK1/2, ERK1/2, β-tubulin and IKKα/β antibodies were purchased from Santa Cruz (Santa Cruz, CA). All other antibodies were obtained from Cell Signaling Technology (Danvers, MA). Cell culture reagents were purchased from Gibco (Shanghai, China).

Li P., Wu Y., Li M., Qiu X., Bai X, & Zhao X. (2015). AS-703026 Inhibits LPS-Induced TNFα Production through MEK/ERK Dependent and Independent Mechanisms. PLoS ONE, 10(9), e0137107.

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Western Blot Analysis of NF-κB Pathway

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Cell pellets were lysed in RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 12 mM deoxycholic acid, 0.5% Nonidet P-40, and protease inhibitors). Protein samples were used for SDS-PAGE followed by Western blotting as previously described (28 (link)). Nitrocellulose membranes were stained with primary antibodies against Tubulin (1:1,000), p-NFκB (p65) (Ser536) (1:1,000), NFκB (p65) (1:1,000), NFκB (p50) (1:1,000); IKB-α (1:500), IKK-α/β (1:500), p-IKB-α (1:500), p-IKK-α/β (1:500) LDH (1:1,000), Sp1 (1:500), TNF-α (1:500), p-p38 (1:1,000), p-ERK1/2 (1:1,000) (Santa Cruz Biotechnology). The nitrocellulose membranes were incubated with the appropriate horseradish peroxidase conjugated secondary antibody (Bio-Rad), and immunoreactive bands were detected by a Fluorchem Imaging System upon staining with ECL Select Western Blotting Detection Reagent (GE Healthcare, Pittsburgh, PA, USA; RPN2235). The Western blots reported are from one experiment out of three separated experiments that gave similar results.
Proteins were assayed by the method described by Lowry et al. (33 (link)).

Limongi D., Baldelli S., Checconi P., Marcocci M.E., De Chiara G., Fraternale A., Magnani M., Ciriolo M.R, & Palamara A.T. (2019). GSH-C4 Acts as Anti-inflammatory Drug in Different Models of Canonical and Cell Autonomous Inflammation Through NFκB Inhibition. Frontiers in Immunology, 10, 155.

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Signaling Pathways Modulation by CCN6

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We purchased p85α, Akt1, p-IKKα/β (Ser180/Ser181), p-IκBα, p65 (Ser536), IKKα/β, IκBα, p65, CCN6, and β-actin primary antibodies (Santa Cruz Biotechnology, CA, USA), and rabbit polyclonal antibodies specific for p-p85 (Y458), p-Akt (S473), p-mTOR (Ser2448), and mTOR (Cell Signaling Technology, Danvers, MA, USA). Recombinant human CCN6 was purchased from PerpoTech (Rocky Hill, NJ, USA) and CCN6 short hairpin (sh)RNA expression plasmids from RNAiCore (Taipei, Taiwan). The D-Luciferin potassium salt was purchased from Gold Biotechnology (St. Louis, MO, USA). Lipofectamine 2000 and TRIzol were purchased from Life Technologies (Carlsbad, CA, USA). Dharmacon Research (Lafayette, CO, USA) supplied ON-TARGETplus siRNAs. Gibco-BRL life technologies (Grand Island, NY, USA) supplied DMEM, α-MEM, fetal bovine serum (FBS), and all other cell culture reagents. The Matrigel was purchased from BD (Biosciences, Bedford, MA, USA) and Promega (Madison, WI,) supplied the pSV-β-Galactosidase Vector and luciferase assay kits. All other USA chemicals or inhibitors that we used were supplied by Sigma-Aldrich (St. Louis, MO, USA).

Tzeng H.E., Tang C.H., Wu S.H., Chen H.T., Fong Y.C., Lu Y.C., Chen W.C., Huang H.D., Lin C.Y, & Wang S.W. (2018). CCN6-mediated MMP-9 activation enhances metastatic potential of human chondrosarcoma. Cell Death & Disease, 9(10), 955.

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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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Molecular Profiling of Inflammatory Signaling

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Anti‐mouse and anti‐rabbit IgG‐conjugated horseradish peroxidase, rabbit polyclonal antibodies specific for PLCβ3 (Santa Cruz, sc‐403), PKCα(Santa Cruz, sc‐208), c‐Src (cell signaling, #2109s), p65 (Santa Cruz, sc‐109), IκBα (Santa Cruz, sc‐1643), p‐PLCβ (cell signaling, #2481s), p‐PKCα(cell signaling, #9375), p‐c‐Src (cell signaling, #5473), p‐p65 (cell signaling, #3033), p‐IKKα/β (cell signaling, #2694), IKKα/β (Santa Cruz, sc‐7218), p‐IκBα (cell signaling, #9246), CXCL13 (GeneTex, GTX108471), CXCR5 (GeneTex, GTX100351), VCAM‐1 (GeneTex, GTX110684) and β‐Actin (GeneTex, GTX109639) were purchased from Cell Signaling Technology, Inc, Santa Cruz Biotechnology, Inc and GeneTex Inc. All other chemicals were obtained from Sigma‐Aldrich. The CXCL13 shRNA plasmid was purchase from RNAiCore (Clone ID: TRCN0000057983). All siRNAs were purchased from Sigma‐Aldrich (Mission^® siRNA).

Chao C., Lee W., Wang S., Chen P., Yamamoto A., Chang T., Weng S, & Liu J. (2021). CXC chemokine ligand‐13 promotes metastasis via CXCR5‐dependent signaling pathway in non‐small cell lung cancer. Journal of Cellular and Molecular Medicine, 25(19), 9128-9140.

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