P ikbα

Manufactured by Santa Cruz Biotechnology

Sourced in Germany, United States

P-IKBα is a laboratory product offered by Santa Cruz Biotechnology. It is a phosphorylated form of the IKBα protein, which is an inhibitor of the NF-κB transcription factor. P-IKBα is used in research applications to study the regulation and signaling pathways involving the NF-κB system.

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Lab products found in correlation

β actin, by Santa Cruz Biotechnology (3 mentions) P ikkα β, by Santa Cruz Biotechnology (2 mentions) Ikkα β, by Santa Cruz Biotechnology (2 mentions) Primary antibody against bcl 2, by Santa Cruz Biotechnology (1 mentions) Pierce ecl western blotting detection reagent, by Thermo Fisher Scientific (1 mentions) Micro bca protein assay kit, by Thermo Fisher Scientific (1 mentions) Quantity one software, by Bio-Rad (1 mentions) Pvdf membrane, by Bio-Rad (1 mentions) Cocktail of protein inhibitors, by Roche (1 mentions) β actin antibody, by Santa Cruz Biotechnology (1 mentions) P akt, by Santa Cruz Biotechnology (1 mentions) Id image analysis software, by Kodak (1 mentions) Horseradish peroxidase hrp conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions) Cd206, by Santa Cruz Biotechnology (1 mentions) Iblot dry blotting system a, by Thermo Fisher Scientific (1 mentions) Protease inhibitor, by Roche (1 mentions) Nitrocellulose membrane, by Cytiva (1 mentions) Bca assay, by Beyotime (1 mentions) Whatman, by Cytiva (1 mentions) Bcl2 associated x (bax), by Cell Signaling Technology (1 mentions)

8 protocols using p ikbα

Western Blot Analysis of Kidney Proteins

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Frozen kidney tissues were homogenized in RIPA buffer containing cocktail of protein inhibitors (Roche, Indianapolis, IN). The samples were centrifuged at 13,000×g for 10 min and supernatant was recovered for the Western blot analysis as described previously (18 (link)). In brief, protein concentrations were determined using the micro BCA protein assay kit (Thermo Scientific, Rockford, IL). Protein samples (80 µg/sample) were subjected to separate by reducing 12% SDS-polyacrylamide gel electrophoresis, and then transferred onto PVDF membrane at 4°C overnight (Bio-Rad, Hercules, USA). The membrane was blocked in 3% non-fat milk in Tris-buffered saline, and probed using a primary antibody against Bcl-2 (Santa Cruz Biotechnology, Dallas, Texas), or caspase-3 (Santa Cruz Biotechnology, Dallas, Texas), or NF-κB p65 (Santa Cruz Biotechnology, Dallas, Texas), or p-IkB-α (Santa Cruz Biotechnology, Dallas, Texas), and subsequently incubated with a secondary HRP-conjugated antibody (Bio-Rad, Hercules, CA). Bands were detected using Pierce ECL Western Blotting detection reagent (Thermo Scientific, Rockford, IL) and the blots were exposed to X-film. The membrane was stripped and reprobed with β-actin antibody (Santa Cruz Biotechnology, Dallas, Texas). The bands on the films were quantified by the Quantity One software (Bio-Rad, Hercules, CA).

Liu J., Abdel-Razek O., Liu Z., Hu F., Zhou Q., Cooney R.N, & Wang G. (2015). Role of surfactant proteins A and D in sepsis-induced acute kidney injury. Shock (Augusta, Ga.), 43(1), 31-38.

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Western Blot Protein Analysis

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The proteins resolved by electrophoresis were transferred from the gel to nitrocellulose membranes using iBlot Dry Blotting System A (Life-Technologies). Membranes were then blocked with PBS (pH 7.2, containing 0.1% (v/v) Tween 20 and 5% (w/v) non-fat dried milk for 1 h, and washed three times with 0.1% Tween 20-PBS (T-PBS) then washed 3 times with 0.1% Tween 20-PBS (T-PBS). Primary antibodies directed against β-actin (1:500), CD40 (1:500), p-Akt (1:500), CD206 (1:500), TLR4 (1:500), and p-IKBα (1:500) (all from Santa Cruz Biotechnology, Inc., Heidelberg, Germany), were incubated for 1 h at room temperature on a shaker and then overnight at 4 °C. At the end, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz Biotechnology), diluted 1:10,000, for 60 min at room temperature in the dark on a shaker after three washes with 0.1% Tween 20- PBS (T-PBS). Chemiluminescence was used to visualize the immunoreactive bands (BioRad Laboratories, Hercules, CA, USA). Densitometric analysis was performed on the bands obtained after immunoblotting using ID Image Analysis Software (Kodak Digital Science). The β-actin was used as a housekeeping protein for normalizing protein expression levels. Results are expressed as arbitrary units.

La Torre M.E., Panaro M.A., Ruggiero M., Polito R., Cianciulli A., Filannino F.M., Lofrumento D.D., Antonucci L., Benameur T., Monda V., Monda M., Porro C, & Messina G. (2022). Extracellular Vesicles Cargo in Modulating Microglia Functional Responses. Biology, 11(10), 1426.

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NF-κB Signaling Pathway Analysis

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Cells were washed with PBS and then lysed in a lysis buffer containing protease inhibitor (Roche, Basel, Switzerland). The concentration of total protein was assessed using BCA assay (Beyotime, Haimen, China). Equal amounts of protein samples were then separated on SDS-PAGE gels and transferred to a nitrocellulose membrane (Whatman, Dassel, Germany). After blocking with 5% skim milk for 1 h, the membranes were probed by primary antibodies against NF-κB, p-NF-κB, p65, p-p65 (Mannheim, Germany), P-IKBα, IKK, IKKα, IKKβ, and P-IKKα/β (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C overnight, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h. After being washed, protein bands were finally visualized with the WEST-ZOL plus Western Blot Detection System.

Huang L., Li F., Deng P, & Hu C. (2016). MicroRNA-223 Promotes Tumor Progression in Lung Cancer A549 Cells via Activation of the NF-κB Signaling Pathway. Oncology Research, 24(6), 405-413.

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Western Blot Analysis of Cell Signaling

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The real-time PCR method has been described in a previous paper [27 (link)]. The primers are presented in Table 1. The cells or mouse colon tissues were lysed for protein immunoblotting, as described previously [28 (link)]. The membrane was probed with antibodies (1 : 1000 dilution) including PI3K p85, p-PI3K p85, Akt, p-Akt, mTOR, p-mTOR, Bcl-2, Mcl-1, Bax (Cell Signaling Technology, Danvers, MA, USA), PHLPP1, PML, MPO, p-IKBα, β-actin, cleaved caspase-3, Foxp3 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), PP2Aa, PP2Ab, and PP2Ac (Protech, Wuhan, China). The intensity of each band was analyzed using the densitometry feature in the ImageJ software (http://rsb.info.nih.gov/ij/).

Fu X., Sun F., Wang F., Zhang J., Zheng B., Zhong J., Yue T., Zheng X., Xu J.F, & Wang C.Y. (2017). Aloperine Protects Mice against DSS-Induced Colitis by PP2A-Mediated PI3K/Akt/mTOR Signaling Suppression. Mediators of Inflammation, 2017, 5706152.

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Signaling Pathway Antibody Toolkit

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Rabbit polyclonal antibodies towards β-actin, PDGF receptor (PDGFR) β, p-PDGFRβ, VEGF, VEGF receptor (VEGFR) 2, p-VEGFR2, nuclear factor kappa B (NF-κB), phosphorylated nuclear factor κB (p-NF-κB), inhibitor of NF-κB (IKB) α and p- IKB α were purchased from Santa Cruz Biotechnology (USA). Pentobarbital sodium and horseradish peroxidase-conjugated secondary goat anti-rabbit antibody were purchased from Sigma (Sigma Chemical Co., St. Louis, MO, USA).

Li W., Jiang Y.X., Chen Q.Y, & Wang G.G. (2021). Recombinant fusion protein by lysozyme and antibacterial peptide enhances ischemic wound healing via angiogenesis and reduction of inflammation in diabetic db/db mice. PeerJ, 9, e11256.

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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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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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Neuroinflammation and Gut Barrier Regulation

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Dgal and donepezil (Done) were purchased from Sangon Biotech Co., Ltd., Shanghai, China and Sigma-Aldrich Co., St. Louis, MO, United States, respectively. Antibiotics (vancomycin, neomycin, ampicillin, and metronidazole) and 4′,6-diamidino-2-phenylindole (DAPI) were obtained from Macklin Shanghai, China. Tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), IL-6, lipopolysaccharide (LPS), and ELISA kits were provided by 4A Biotech, Beijing, China and Hefei Laier Biotech, Anhui, China. The antibodies of TLR4 (4A Biotech, Beijing China), occludin, pIKBα, zonulin (Santa Cruz Biotechnology, Dallas, Texas, United States), neuronal nuclei (NeuN), IKBα, Iba-1 (Cell Signaling Technology, Boston, United States), GADPH, secondary antibody (horseradish peroxidase [HRP]–linked anti-rabbit and anti-mouse IgGs), and ECL Western blot chemiluminescence detection kit (Beijing CoWin Biotech Company, Beijing, China) were used in the present study. All primers in this work were synthesized by Sangon Biotech Co., Ltd., Shanghai, China.

Fasina O.B., Wang J., Mo J., Osada H., Ohno H., Pan W., Xiang L, & Qi J. (2022). Gastrodin From Gastrodia elata Enhances Cognitive Function and Neuroprotection of AD Mice via the Regulation of Gut Microbiota Composition and Inhibition of Neuron Inflammation. Frontiers in Pharmacology, 13, 814271.

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