Anti nf κb

Manufactured by Cell Signaling Technology

Sourced in United States, United Kingdom, China

Anti-NF-κB is a laboratory reagent used for the detection and analysis of the NF-κB transcription factor in biological samples. NF-κB is a key regulator of various cellular processes, including immune response, inflammation, and cell survival.

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NF-κB (393 citations) Anti-NF-κB p65 (D14E12, (10 citations) Anti-NF-κB p65 rabbit monoclonal antibody (4 citations) Rabbit anti-mouse phospho-NF-κB p65 antibody (2 citations) Rabbit anti-mouse NF-κB p65 (2 citations) Anti-p-NF-κB-p65 antibody (2 citations) Anti-nuclear factor kappa B (NF-κB) (2 citations) Anti-NF-κB p65 monoclonal (D14E12) (2 citations) Rabbit monoclonal anti-NF-κB/RelA (2 citations) Anti-NF-κB p65 clone D14E12 (2 citations)

140 protocols using anti nf κb

Investigating SYK and NF-κB Signaling

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Unless stated otherwise, all chemicals were purchased from MilliporeSigma. Antibodies were obtained from the following sources: anti–p-Tyr (catalog 9411), anti–p-SRC (Y416) (catalog 2101), anti-SRC (catalog 2109), anti–p-SYK (Y525/526) (catalog 2710), anti-SYK (catalog 2712), anti–p-IκBα (catalog 2859), anti-IκBα (catalog 9242), anti-GAPDH (catalog 2118), anti-TLR2 (catalog 12276 for human and 13744 for mouse), anti–NF-κB (catalog 3033), and anti–NF-κB (catalog 8242) from Cell Signaling Technology; anti-MyD88 (catalog SAB3500472) and anti-FLAG M2 (catalog F3165) from MilliporeSigma; anti-Myc (catalog sc-40) and anti-actin (catalog sc-47778) from Santa Cruz Biotechnology; anti-HA (M180-3) from MBL International; anti–p-Tyr (4G10) (catalog 05-321) from Millipore; anti-PPP1R11 (ab171960) from Abcam; and anti-3BP2 (catalog H00006452-M01) from Abnova. Halt Protease and Phosphatase Inhibitor Cocktail was from Thermo Fisher Scientific. SYK inhibitor was from Millipore. PP2 and BMS 345541 were from Selleckchem.

Matsumoto Y., Dimitriou I.D., La Rose J., Lim M., Camilleri S., Law N., Adissu H.A., Tong J., Moran M.F., Chruscinski A., He F., Asano Y., Katsuyama T., Sada K.E., Wada J, & Rottapel R. (2022). Tankyrase represses autoinflammation through the attenuation of TLR2 signaling. The Journal of Clinical Investigation, 132(7), e140869.

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Protein Expression Analysis in HBMECs

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HBMECs were plated on T75 flasks and treated for 24 h. At the end of the treatment period, cells were washed twice with ice-cold phosphate-buffered saline (PBS) and harvested into lysis buffer (Thermo Fisher # 89900) containing protease inhibitors (Thermo Fisher # 78442). The protein content in each sample was determined using BCA Assay (Thermo Fisher # 23225). Equal amounts of protein (30 μg) were loaded in each well and run on SDS–PAGE. The separated proteins were then transferred onto nitrocellulose membranes using Power Blotter XL (Thermo Fisher # PB0013). 5 % nonfat milk in Tris-buffered saline containing 0.1 % Tween-20 was used to block the membranes for 45 min at room temperature. The membranes were then incubated with anti-NRF2 antibody (1:500) (Santa Cruz # sc-365949), anti-NF-κB (1:1000) (Cell Signaling # 8242S), anti-ZO-1 (1:1000) (Invitrogen # 61–7300), anti-Occludin (Invitrogen # 33–1500) (1:1000) anti-β-Actin antibody (1:1000), anti-GAPDH antibody (1:5000) (Cell Signaling # sc-47724) at 4 °C overnight, followed by washing and incubation in HRP-conjugated secondary antibodies for one hour (1:10,000). Immuno-reactive bands were visualized using a chemiluminescence system according to the manufacturer’s instructions. Band densities were analyzed using ImageJ and expressed as fold change over controls after normalizing them against β-actin and GAPDH.

Doleman B.J., Severin E.J, & Lewis N.S. (1998). Trends in odor intensity for human and electronic noses: Relative roles of odorant vapor pressure vs. molecularly specific odorant binding. Proceedings of the National Academy of Sciences of the United States of America, 95(10), 5442-5447.

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Western Blot Analysis of Signaling Pathways

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B16-F10 cells (2 × 10⁶ cells per well) were seeded into a 10 cm dish for 24 h, and then cells were treated with different concentrations of PSS (12.5, 25, 50, 100 μg/mL) for 24 h. The medium was removed, and the cells were washed with PBS three times. Cells were then lysed in 200 μL of lysis buffer on ice. The total protein was determined using the Bicinchoninic Acid (BCA) Kit (Solarbio, Beijing, China). Equal amounts of protein in the cell extracts were fractionated by 10% SDS-PAGE, and then electrotransferred onto polyvinylidene fluoride (PVDF) membranes. After blocking with TBST (20 mM Tris-buffered saline and 0.1% Tween) containing 5% nonfat dry milk for 1 h at room temperature, the membranes were incubated for 2 h with monoclonal antibodies, such as anti-MMP-9, anti-MMP-2, anti-E-cadherin, anti-Vimentin, anti-ERK 1/2, anti-p-ERK 1/2, anti-AKT, anti-p-AKT (Ser473), anti-p38, anti-p-p38, anti-p-p38, anti-NF-κB, anti-p-NF-κB, and anti-β-actin, which were purchased from Cell Signaling Technology. The membranes were then washed three times and incubated with HRP-conjugated secondary antibodies (Abcam, Cambridge, Massachusetts, USA). The proteins were then detected using chemiluminescence agents (Amersham ECL, GE Healthcare, Buckinghamshire, UK).

Ma H., Qiu P., Xu H., Xu X., Xin M., Chu Y., Guan H., Li C, & Yang J. (2019). The Inhibitory Effect of Propylene Glycol Alginate Sodium Sulfate on Fibroblast Growth Factor 2-Mediated Angiogenesis and Invasion in Murine Melanoma B16-F10 Cells In Vitro. Marine Drugs, 17(5), 257.

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Western Blot Analysis of Signaling Proteins

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Western blot analysis was performed as described previously [33 (link)] with minor modifications. Briefly, aliquots of 30–60 µg of protein were subjected to 8–12% SDS-PAGE and transferred onto Hybond-P⁺ polyvinylidene difluoride membranes (Amersham, Buckinghamshire, UK). The membranes were incubated with the following primary antibodies: anti-ICAM-1 (ab225884, 1:1000, Abcam, Cambridge, UK), anti-VCAM-1 (ab106778, 1:1000, Abcam), anti-phospho-ERK (sc-7383, 1:1000, Santa Cruz Biotechnology), anti-total-ERK (sc-94 1:1000, Santa Cruz Biotechnology), anti-phospho-PKC (9375S, 1:1000, Cell Signaling Technology, Danvers, MA, USA), anti-PKC (sc-10800, 1:1000, Santa Cruz Biotechnology), anti-phospho-PDK1 (3061S, 1:1000, Cell Signaling Technology), anti-PDK1 (3062S, 1:1000, Cell Signaling Technology), anti-phospho-STAT-3 (9131S, 1:1000, Cell Signaling Technology, anti-STAT-3 (4904S, 1:1000, Cell Signaling Technology), anti-HIF-1α (ab2185, 1:1000, Abcam), anti-phospho-NF-κB (8242S, 1:1000, Cell Signaling Technology), anti-NF-κB (8242S 1:1000, Cell Signaling Technology), and anti-β-actin (A2066, 1:2000, Sigma-Aldrich, St. Louis, MO, USA). The bound antibodies were detected with horseradish peroxidase-conjugated secondary antibodies and an ECL western blotting detection reagent (Bio-Rad, Hercules, CA, USA).

Jin H., Rugira T., Ko Y.S., Park S.W., Yun S.P, & Kim H.J. (2020). ESM-1 Overexpression is Involved in Increased Tumorigenesis of Radiotherapy-Resistant Breast Cancer Cells. Cancers, 12(6), 1363.

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Detailed Immunological Antibody Protocols

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The antibodies used in this study for flow cytometry include: anti-mouse CD8 (#563786), anti-mouse CD25 (#558642), anti-mouse TCRβ (#553174), anti-human CD3 (#563798), anti-human CD8 (#563795), and anti-human TCRαβ (#563826) from BD Biosciences; anti-mouse CD4 (#100544), anti-mouse IFN-γ (#505835), anti-human LFA-1 (#363404), anti-human CD11a (#301208), anti-human CD18 (#302114), anti-human CD25 (#302625), and anti-human CD69 (#310920) from BioLegend; anti-mouse CD4 (#17-0042-83), anti-mouse CD4 (#17-0041-83), anti-mouse CD69 (#25-0691-82), anti-mouse TNF (#17-7321-82), anti-mouse IL-2 (#12-7021-82), anti-human CD4 (#17-0048-2), and anti-human CD8 (#11-0088-41) from eBioscience.
The antibodies used for immunoprecipitation and immunoblotting include: anti-Cdc7 (#ab10535), anti-Dbf4 (#ab124707), anti-RNA polymerase II (#ab817) and anti-RNA polymerase II (phospho S2, # ab193468) from Abcam; anti-PLCγ (#610027) and anti-ERK (#610031) from BD Biosciences; anti-LAT (#641102) from BioLegend; anti-GAPDH (#2118), anti-pPLCγ (#2821), anti-ZAP70 (#2709), anti-pZAP70 (#2717), anti-pERK (#4370), anti-pLAT (#3584), anti-pLck (#6943), and anti-NF-κB (#3035) from Cell Signaling; anti pMCM2 S40 (#GTX62847) from GeneTex; anti-MCM2 (#MA5-15895) from Pierce; and anti-Lck (#sc-433) from Santa Cruz.

Chen E.W., Tay N.Q., Brzostek J., Gascoigne N.R, & Rybakin V. (2019). A Dual Inhibitor of Cdc7/Cdk9 Potently Suppresses T Cell Activation. Frontiers in Immunology, 10, 1718.

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Western Blot Analysis of Key Signaling Proteins

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Cells were lysed with an NP-40 buffer containing protease inhibitor cocktail (Roche, 11697498001). Protein (20 μg) was resolved by 4–15% SDS-polyacrylamide gel electrophoresis (Bio-Rad). After transfer, polyvinylidene difluoride membranes were blotted with anti-HIF-1α (1 : 1000, Cell Signaling, 14179), anti-Stat3 (1 : 1000, Cell Signaling, 12640), anti-NF-κB (1 : 1000, Cell Signaling, 4882), anti-CD40 (1 : 1000, Cell Signaling, 86165), or anti-GAPDH (1 : 3000, Cell Signaling, 5174) antibody overnight at 4 °C. Proteins were detected with goat anti-rabbit or anti-mouse IgG-HRP conjugate (1 : 5000, Bio-Rad, 1706515 or 1706516), followed by ECL development (GE Healthcare, RPN2232). Full scan images of blots are available as a Source data file.

Yang F., He Z., Duan H., Zhang D., Li J., Yang H., Dorsey J.F., Zou W., Nabavizadeh S.A., Bagley S.J., Abdullah K., Brem S., Zhang L., Xu X., Byrne K.T., Vonderheide R.H., Gong Y, & Fan Y. (2021). Synergistic immunotherapy of glioblastoma by dual targeting of IL-6 and CD40. Nature Communications, 12, 3424.

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Western Blot Analysis of Inflammatory Signaling

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Cells were lysed and sonicated in radioimmunoprecipitation (RIPA) buffer with protease inhibitor mixture, PMSF, and sodium orthovanadate. Protein concentration was measured by BCA protein assay (Thermo scientific, Rockford, IL, USA). The samples were resolved by SDS-PAGE and transferred to nitrocellulose membrane and blotted with specific antibodies: anti-p58^IPK, anti-p-NF-κB, anti-NF-κB, anti-p-p38, anti-p38, anti-p-JNK, anti-JNK, (Cell Signaling Technology, Boston, MA, USA), anti-p-PKR, anti-PKR, anti-β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-IL-1β (R&D Systems, Inc., Minneapolis, MN, USA), anti-caspase-1 (p-20), and anti-NLRP3 (AdipoGen, Inc, San Diego, CA, USA). The immunoblots were developed using chemiluminescence (SuperSignal West Dura Extended Duration Substrate, Thermo Scientific, USA) and visualized under Chemidoc MP Imaging System (Bio-Rad, Hercules, CA, USA).

Boriushkin E., Wang J.J., Li J., Bhatta M, & Zhang S.X. (2016). p58IPK suppresses NLRP3 inflammasome activation and IL-1β production via inhibition of PKR in macrophages. Scientific Reports, 6, 25013.

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Comprehensive Immunoblot Analysis of Inflammatory Signaling

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The following antibodies (Abs) and reagents were used in present studies: anti-IL-1β (human specific, mouse specific, and human & mouse specific), anti-phospho NF-κB, anti-NF-κB, anti-phospho ERK, anti-ERK, anti-phospho p38, anti-p38, anti-phospho 4E-BP1, anti-4E-BP1, anti-phospho Mnk1, anti-Mnk1, anti-phospho eIF4E, anti-eIF4E, anti-phospho MK2, anti-MK2, and anti-streptavidin-HRP antibodies were purchased from cell signaling technology (Danvers, MA). Anti-β-actin, Dimethyl sulfoxide, Actinomycin D, Cyclohexamide, ultrapure E. coli O111:B4 LPS, Rapamycin, PD98059, and SB202190 were purchased from Sigma-Aldrich (St. Louis, MO). Anti-NLRP3 antibody was purchased from Adipogen (San Diego, CA). Torin 1 and FR180204 were purchased from Merck Millipore (Billerica, MA). CGP57380 and MK25 were purchased from TOCRIS (Ellisville, MO, USA) and Cayman Chemical (Ann Arbor, MI), respectively. Recombinant human M-CSF and mouse M-CSF were purchased from R&D system (Minneapolis, MN).

Chung Y.H., Kim D.H, & Lee W.W. (2016). Monosodium urate crystal-induced pro-interleukin-1β production is post-transcriptionally regulated via the p38 signaling pathway in human monocytes. Scientific Reports, 6, 34533.

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Protein Expression Analysis in Rat Tissues

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The different rat tissues were homogenized and lysed. The adipocytes were harvested and lysed. Equal amounts of proteins were boiled and separated by SDS-PAGE and transferred onto nitrocellulose membranes. The following primary antibodies were used: anti-AAT1 (Sigma-Aldrich, St. Louis, MO, USA), anti-AAT2 and anti-GAPDH (Kangcheng, Shanghai, China), anti-p-NF-κB, anti-NF-κB, anti-p-IκBα and anti-IκBα (Cell Signaling Technology, Danvers, MA, USA). HRP-labeled Sigma-Aldrich goat anti-mouse and Sigma-Aldrich goat anti-rabbit at dilutions of 1:6000 and 1:4000 were used as secondary antibodies. The bands were visualized using Enhanced Chemiluminescence Detection Kit (Thermo Scientific, Rockford, IL, USA). The densitometric analysis of the positive bands was performed by use of AlphaImager (San leandro, CA, USA).

Zhang H., Huang Y., Bu D., Chen S., Tang C., Wang G., Du J, & Jin H. (2016). Endogenous sulfur dioxide is a novel adipocyte-derived inflammatory inhibitor. Scientific Reports, 6, 27026.

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Molecular Mechanisms in Neurological Injury

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The tissue samples from the striatum and cortex of the ipsilateral hemisphere were homogenized in a buffer consisting of 20 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton, 1 mM Na2EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, and protease inhibitors. Equal amounts of total protein from each group underwent immunoblotting assays. Proteins were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunoblotting with anti-HMGB1, anti-phospho-inducible nitric oxide synthase (iNOS), anti-iNOS, anti-phospho-endothelial NOS (eNOS), anti-eNOS (Abcam, Cambridge), anti-Toll-like receptor-4 (TLR-4), anti-Actin (Santa Cruz, CA), anti-Bcl-2, anti-Bax, anti-cytochrome C, anti-cleaved caspase-3, anti-caspase-3, anti-phospho-nuclear factor-kappa B (NF-κB), and anti-NF-κB, (all Cell Signaling Technology, Beverly, MA) was performed at least three times each.

Song Y., Jun J.H., Shin E.J., Kwak Y.L., Shin J.S, & Shim J.K. (2017). Effect of pregabalin administration upon reperfusion in a rat model of hyperglycemic stroke: Mechanistic insights associated with high-mobility group box 1. PLoS ONE, 12(2), e0171147.

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