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P ikb

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P-IkB is an antibody product developed by Cell Signaling Technology. It is a phospho-specific antibody that detects IκB-α protein phosphorylated at Ser32 and Ser36. IκB-α is a key regulator of the NF-κB signaling pathway.

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Lab products found in correlation

β actin, by Cell Signaling Technology (4 mentions) P jnk, by Cell Signaling Technology (3 mentions) P c jun, by Cell Signaling Technology (3 mentions) P akt, by Cell Signaling Technology (2 mentions) Histone 3, by Cell Signaling Technology (2 mentions) β actin, by Merck Group (2 mentions) Supersignal west femto, by Thermo Fisher Scientific (2 mentions) Lipopolysaccharides (lps), by Merck Group (2 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions) Anti nampt, by Fortis Life Sciences (1 mentions) Nucleolin, by Cell Signaling Technology (1 mentions) Anti mcl 1, by Santa Cruz Biotechnology (1 mentions) Penicillin streptomycin, by Welgene (1 mentions) Trizol reagent, by Thermo Fisher Scientific (1 mentions) Hrp conjugated secondary antibody, by Cell Signaling Technology (1 mentions) Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions) Dimethyl sulfoxide (dmso), by Merck Group (1 mentions) Cox 2, by Cell Signaling Technology (1 mentions) Pro prep, by iNtRON Biotechnology (1 mentions) P nf κb, by Cell Signaling Technology (1 mentions)

Spelling variants of this product

P-IkB- α(ser32 (3 citations)

16 protocols using p ikb

1

Western Blot Analysis of Signaling Proteins

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WM cells were cultured with or without stimuli, harvested, washed, and lysed using radioimmunoprecipitation assay buffer. Proteins were separated on an SDS-polyacrylamide gel and electroblotted on a polyvinylidene difluoride (PVDF) membrane (Pall Gelman Laboratory, Ann Arbor, MI). Proteins were visualized by probing the membranes with the following antibodies: anti-tubulin, -caspase-3, -PARP, p44/42 MAPK (ERK 1/2), p-p44/42 MAPK, p-AKT(Ser473), AKT, p-GSK-3α/β (Ser21/9), GSK-3α/β, Bruton's tyrosine kinase (BTK), pBTK (Tyr223), -p-p65, -p-p52, -RelB, -p-IkB, -nucleolin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Cell Signalling Technology); anti-Nampt (Bethyl Laboratories); anti-Mcl-1 (Santa Cruz Biotechnology). Standard enhanced chemiluminescence was used for protein band detection.
Cea M., Cagnetta A., Acharya C., Acharya P., Tai Y.T., Yang G., Lovera D., Soncini D., Miglino M., Fraternali-Orcioni G., Mastracci L., Nencioni A., Montecucco F., Ballestrero A., Hideshima T., Chauhan D., Gobbi M., Lemoli R.M., Munshi N., Treon S.P, & Anderson K.C. (2016). Dual NAMPT and BTK Targeting Leads to Synergistic Killing of Waldenstrom's Macroglobulinemia Cells Regardless of MYD88 and CXCR4 Somatic Mutations Status. Clinical cancer research : an official journal of the American Association for Cancer Research, 22(24), 6099-6109.
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2

Anti-inflammatory Activity Evaluation

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The following chemicals and reagents were utilized: Roswell Park Memorial Institute Medium (RPMI), fetal bovine serum (FBS), penicillin-streptomycin, and Dulbecco's phosphate buffered saline (DPBS) (Welgene, South Korea); CFA and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich, St. Louis, MO, USA); Oligo-dT (Bioneer oligo synthesis); COX-2, iNOS, TNF-α, IL-1β, and IL-6 primers (Bioneer, Daejeon, South Korea); dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO, USA); TRIzol reagent (Invitrogen, Carlsbad, CA, USA); Pro-prep (iNtRON biotechnology, South Korea); bovine serum albumin (BSA) (Thermo Fisher Scientific, South Korea); specific antibodies used for western blot, including COX-2, iNOS, p-IKB, p-NF-κB, β-actin, and HRP-conjugated secondary antibody (Cell Signaling Technology, Danvers, MA, USA); secondary antibody used for immunofluorescence (anti-rabbit IgG Fab2, Alexa Fluor 555, Molecular Probes). All other chemicals and reagents were the highest quality available.
Ullah H.M., Lee Y.Y., Kim S.D, & Rhee M.H. (2021). Duchesnea indica Extract Attenuates Coal Fly Ash-Induced Inflammation in Murine Alveolar Macrophages through the NF-Kappa B Pathway. Evidence-based Complementary and Alternative Medicine : eCAM, 2021, 5546052.
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3

Western Blot Analysis of Protein Expression

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Proteins were separated by SDS-PAGE, transferred to polyvinylidene difluoride membrane (EMD Millipore, Billerica, MA, USA), and incubated overnight at 4°C with antibody against TRIM32 (1:700; Santa Cruz Biotechnology Inc.), p-IkB, IkB, p-AKT, p-ERK Bcl-2, cytochrome c, caspase 3, cleaved caspase 3 (1:1,000; Cell Signaling Technology, Danvers, MA, USA), and GAPDH (1:2,000; Santa Cruz Biotechnology Inc.). After incubation with HRP-linked anti-mouse/rabbit IgG (1:2,000; Santa Cruz Biotechnology Inc.) at 37°C for 2 hours, visualization was performed using enhanced chemiluminescence (Thermo Fisher Scientific) and DNR BioImaging Systems (DNR, Neve Yamin, Israel).
Du Y., Zhang W., Du B., Zang S., Wang X., Mao X, & Hu Z. (2018). TRIM32 overexpression improves chemoresistance through regulation of mitochondrial function in non-small-cell lung cancers. OncoTargets and therapy, 11, 7841-7852.
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4

Glioblastoma Cell Line Culture and Analysis

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Human GBM cell lines (U87, U251, A172, T98G, LN229 and LN18) used in this study were cultured and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% foetal bovine serum (FBS). GSC2 was cultured in neurobasal medium containing basic fibroblast growth factor, epidermal growth factor, B27 supplement, harpin, l‐glutamine and N2 supplement.26 Primary antibodies for Csnk1a1, β‐actin, P65, p‐P65, p‐IkB and IkB were purchased from Cell Signaling Technology (CST, Beverly, MA, USA). p‐FOXO1a (S322/S325) and FOXO1a were purchased from Abcam (Cambridge, MA, USA). Csnk1a1 inhibitor D4476 was purchased from Selleck Chemicals (Houston, TX, USA). D4476 was dissolved in DMSO to create a 10 mmol/L solution, which was diluted to different concentrations before use.
Liu G., Li H., Zhang W., Yu J., Zhang X., Wu R., Niu M., Liu X, & Yu R. (2021). Csnk1a1 inhibition modulates the inflammatory secretome and enhances response to radiotherapy in glioma. Journal of Cellular and Molecular Medicine, 25(15), 7395-7406.
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5

Western Blot Analysis of NF-κB Pathway

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WB was performed as per our previous protocol [42 (link)]. The corresponding antibodies to the protein of interest for immunoblotting included p-IKKα/β (Cell Signaling Technology, Danvers, MA, USA, 2697, 1:1000 dilution), p-IkB (Cell Signaling Technology, 9246, 1:1000 dilution), IKKα/β (Abcam, Cambridge, MA, USA, ab178870, 1:1000 dilution), IkB (Cell Signaling Technology, 9242, 1:1000 dilution), p65 (BD Bioscience, San Jose, CA, USA, 610868, 1:1000 dilution), NLRC5 (Abcam, ab105411, 1:500 dilution), p-TAK1 (ImmunoWay, Plano, TX, USA, YP1522, 1:500 dilution), TAK1 (ImmunoWay, YT4536, 1:1000 dilution), cleaved-Caspase-1 p20 (Santa Cruz Biotechnology, Santa Cruz, CA, USA, sc1218, 1:1000 dilution), β-actin (Sigma-Aldrich, A5441, 1:5000 dilution), and Histone-3 (Cell Signaling Technology, 4499, 1:2000 dilution). The WB band was analyzed and quantitated by ImageJ 1.6.0 (NIH, Bethesda, MD, USA).
Sun C., Shen Y., Liu P., Shen Y., Hu Y., Li P., Zhang Y., Miao F, & Zhang J. (2023). NLRC5 Deficiency Reduces LPS-Induced Microglial Activation via Inhibition of NF-κB Signaling and Ameliorates Mice’s Depressive-like Behavior. International Journal of Molecular Sciences, 24(17), 13265.
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6

Protein Fractionation and Western Blotting

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A total of 25 µg of protein was fractionated on 4–15% gradient SDS-PAGE gels and transferred to nitrocellulose membranes using the standard protocols. The following primary antibodies were used: p-ERK1/2 (#4370), p-p38 (#4511), p-JNK (#4668), p-IKB (#2859), NF-kB2 p100/p52 (#4882), Fn14 (#4403), p-AktS473 (#9271), FASN (#3180S) and CPT-1A (#12252) and were purchased from Cell Signaling Technology (Danvers, MA, USA), FABP4 (SC18661) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA) and anti-ß-actin was purchased from Sigma-Aldrich. The antibodies were used at the dilutions recommended by the manufacturers.
Altuna-Coy A., Ruiz-Plazas X., Alves-Santiago M., Segarra-Tomás J, & Chacón M.R. (2021). Serum Levels of the Cytokine TWEAK Are Associated with Metabolic Status in Patients with Prostate Cancer and Modulate Cancer Cell Lipid Metabolism In Vitro. Cancers, 13(18), 4688.
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7

Western Blot Assay Protocol

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Cells were lysed in Laemmli sample buffer (Sigma-Aldrich, St. Louis MO) supplemented with protease inhibitor cocktail (Roche Diagnostics, Indianapolis IN). Samples were separated on SDS–PAGE gels (Bio-Rad, Hercules CA) and transferred to nitrocellulose membranes (Bio-Rad, Hercules CA) for protein detection as described. Primary antibodies were obtained from Cell Signaling Technology (Danvers MA): p-IKB (2859s, 1:1000 dilution, p-p65 (3039s, 1:1000 dilution), p-Akt (4046s, 1:2000 dilution), p-38MAPK (4092s, 1:1000 dilution), p-SRC (59548s, 1:1000 dilution), Dpp4 (67138s, 1:1000 dilution). Other antibodies were obtained thus: c-src/src (sc-8056, 1:1000 dilution, Santa Cruz Biotechnologies, Dallas TX), β-actin (sc-47778, 1:2000 dilution, Santa Cruz Biotechnologies, Dallas TX), Flag M2 (F1804, 1:10,000 dilution, Sigma-Aldrich, St. Louis MO), and anti HA (16B12, 1:10,000, Covance, Princeton NJ) as well as horseradish peroxidase (HRP) conjugated secondary antibodies (HAF007, 1:1,000, and HAF005, 1:1,000, R&D system, Minneapolis MN) and chemi-luminescent substrate (Invitrogen, Waltham MA).
Wang C., Nistala R., Cao M., Pan Y., Behrens M., Doll D., Hammer R.D., Nistala P., Chang H.M., Yeh E.T, & Kang X. (2023). Dipeptidylpeptidase 4 promotes survival and stemness of acute myeloid leukemia stem cells. Cell reports, 42(2), 112105.
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8

Protein Expression Analysis Protocol

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The total cellar proteins were separated applying RIPA buffer (Invitrogen, USA) in accordance with manufacture instructions. Then, we evaluated the concentration of proteins by a BCA protein assay kit. We separated 25 μg of each protein samples on 12% SDS-PAGE gels, and then transferred onto the PVDF membranes (BioRad, USA), incubated with 5% no-fat milk, cultured in the primary antibodies against PEG (Abcam, USA, 1:100), COX-2 (1:500), iNOS (1:1000), MMP-1 (1:1000), MMP-3 (1:1000), MMP-13 (Abcam, 1:1000), p-p65 (Cell Signaling Technology, USA, 1:1000), p65 (1:1000), IkB (1:1000), p-IkB (Cell Signaling Technology, 1:1000), and GAPDH (Proteintech, Wuhan, China, 1:5000) overnight at 4 °C, and then blocked at room temperature with secondary antibodies for 2 h. Finally, we used ECL detection system reagents (Millipore, MA, USA) to visualize the protein blots.
Yi H., Zhang W., Cui Z.M., Cui S.Y., Fan J.B., Zhu X.H, & Liu W. (2020). Resveratrol alleviates the interleukin-1β-induced chondrocytes injury through the NF-κB signaling pathway. Journal of Orthopaedic Surgery and Research, 15, 424.
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9

Investigating Cellular Signaling Pathways

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We treated cells in triplicate with 0.1% DMSO, 5 µg/ml LPS (Sigma-Aldrich) or 50 µg/ml RGAP for 6 hours. After electrophoresis of extracted proteins (10 µg/lane), they were transferred to nitrocellulose membranes, which were blocked by 1 hour preparation with 5% skim milk. Rabbit polyclonal antibodies against p-c-jun (1:1,000 dilution; Cell Signaling, Beverly, MA, USA), p-JNK (1:1,000 dilution; Cell Signaling) or p-iKB (1:1,000 dilution; Cell Signaling) were used. The secondary antibody was Horseradish peroxidase-conjugated donkey anti-rabbit Ig (1:7,000; Jackson ImmunoResearch, West Grove, PA, USA). Super signal West Femto (Thermo Scientific, Rockford, IL, USA) presented the bands. Mouse monoclonal antibody of β-actin (1:5,000; Chemicon, Temecula, CA, USA) was used.
Lee H.J., Kwack M.H, & Lee W.J. (2021). Red Ginseng Acidic Polysaccharides Promote the Expression of Acne-Related Inflammatory Biomarkers in Lipopolysaccharide-Treated Sebocytes and Outer Root Sheath Cells and Cutibacterium acnes-Injected Mice. Annals of Dermatology, 33(5), 409-418.
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10

Resveratrol-Enriched Rice Modulates Inflammatory Responses

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Dulbecco's modified Eagle medium (DMEM), fetal bovine serum (FBS), and penicillin-streptomycin were purchased from Invitrogen (Carlsbad, CA, USA). LPS, NG-mono-methyl-L-arginine (L-NMMA), and trans-resveratrol were purchased from Sigma-Aldrich (St. Louis, MO, USA). A normal rice (NR) (Oryza sativa var. japonica) and resveratrol-enriched rice (RR) were supplied by the Rural Development Administration (RDA) of South Korea as mentioned in our previous paper [28 (link)]. Enzyme-linked immunosorbent assay (ELISA) development kits for tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), prostaglandin E2 (PGE2), and IL-1β were purchased from R&D Systems (Minneapolis, MN, USA). Primary and secondary antibodies against inducible nitric oxide synthase (iNOS), cyclooxygenase (COX-2), extracellular signal-regulated kinases (ERK), C-Jun N-terminal kinase (JNK), p38, NF-κB, Histone-3, β-Actin, IkB, pIkB, C-Fos, p-C-Fos, C-Jun, p-C-Jun, and tubulin were purchased from Cell Signaling (Beverly, MA, USA). All other chemicals and reagents were purchased from Sigma Chemical (St. Louis, MO).
Subedi L., Baek S.H, & Kim S.Y. (2018). Genetically Engineered Resveratrol-Enriched Rice Inhibits Neuroinflammation in Lipopolysaccharide-Activated BV2 Microglia Via Downregulating Mitogen-Activated Protein Kinase-Nuclear Factor Kappa B Signaling Pathway. Oxidative Medicine and Cellular Longevity, 2018, 8092713.
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