The cells subjected to various treatments (in 24-well plates) were rinsed thrice with PBS and fixed using 4% cold PBS-buffered paraformaldehyde, followed by permeabilization with 0.2% Triton X-100. The cells were then incubated and blocked with the blocking solution (5% BSA/PBS), followed by incubation overnight at 4°C with the primary antibody—rabbit anti-NF-κB (1:1000, Cell Signaling Technology). Thereafter, the cells were incubated with Alexa Fluor® 488-Conjugated Goat anti-Rabbit IgG (H+L) (1:100,ZSGB-BIO, China) for 1 h in the cassette after washing them with PBS; they were then dyed with 10 μg/mL ready-to-use DAPI solution (Solarbio) for 3 min to stain the nuclei. Representative areas were photographed using a laser scanning confocal microscope (Zeiss, Germany). The experiment was repeated thrice.
Rabbit anti nf κb
Manufactured by Cell Signaling Technology
Sourced in United States
Rabbit anti-NF-κB is a primary antibody that recognizes the NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) protein. NF-κB is a transcription factor that plays a crucial role in the regulation of immune and inflammatory responses. This antibody can be used in various immunodetection techniques to study the expression and localization of NF-κB in biological samples.
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Lab products found in correlation
Rabbit anti akt, by Cell Signaling Technology (2 mentions)
Rabbit anti phospho akt, by Cell Signaling Technology (2 mentions)
Rabbit anti gapdh, by Cell Signaling Technology (2 mentions)
Rabbit anti phospho nf κb, by Cell Signaling Technology (2 mentions)
Confocal laser scanning microscope, by Zeiss (1 mentions)
Alexa fluor 488 conjugated goat anti rabbit igg h l, by ZSGB-BIO (1 mentions)
Dapi solution, by Solarbio (1 mentions)
Supersignal ecl kit, by Thermo Fisher Scientific (1 mentions)
Rabbit anti enos, by Merck Group (1 mentions)
Rabbit anti bax, by Bioworld Technology (1 mentions)
Hrp conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Streptavidin hrp, by Jackson ImmunoResearch (1 mentions)
Mouse anti irf3, by Cell Signaling Technology (1 mentions)
Rabbit anti tbk1, by Cell Signaling Technology (1 mentions)
Rabbit anti ifit1, by Cell Signaling Technology (1 mentions)
Rabbit anti sars cov 2 nucleocapsid, by Cell Signaling Technology (1 mentions)
Species specific hrp conjugated antibodies, by Jackson ImmunoResearch (1 mentions)
Rabbit anti phospho irf3, by Cell Signaling Technology (1 mentions)
Rabbit anti irf3, by Cell Signaling Technology (1 mentions)
16 protocols using rabbit anti nf κb
1
Immunofluorescent Analysis of NF-κB
2
Protein Expression Analysis in Myocardial Tissues
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Total protein was obtained from left ventricular myocardial tissues by sonication, centrifugation and heat denaturation. The protein lysates were electrophoresed and separated on 6–12% SDS-PAGE and transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked with 5% skim milk at room temperature for 1 h, and then incubated overnight at 4°C with primary antibodies, including rabbit anti-Akt (1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), rabbit anti-phospho Akt (1:1,000; Cell Signaling Technology, Inc.), rabbit anti-eNOS (1:1,000; Sigma), rabbit anti-phospho-eNOS (1:200; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), rabbit anti-NF-κB (1:800; Cell Signaling Technology, Inc.), rabbit anti-Bcl-2 (1:800; BioWorld, Inc., Visalia, CA, USA), rabbit anti-Bax (1:800; BioWorld, Inc.), and rabbit anti-GAPDH (1:1,000; Cell Signaling Technology, Inc.). The membranes were then incubated with HRP-conjugated secondary antibodies (1:500; Santa Cruz Biotechnology, Inc.) at room temperature for 1 h. The SuperSignal ECL kit (Thermo Fisher Scientific, Rockville, MD, USA) was used to detect the antigen-antibody complexes in a western blotting detection system (Bio-Rad). The results were expressed as density values normalized to GAPDH.
3
Antibody Profiling for SARS-CoV-2 Analysis
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Antibodies used for immunoblot and immunofluorescence analysis include: rabbit anti-Flag (1:1000, Sigma), mouse anti-Flag M2 (1:1000, Sigma), rabbit anti-IRF3 (1:100 or 1:500, Cell Signaling Technology), mouse anti-IRF3 (1:1000, Cell Signaling Technology) rabbit anti-TBK1 (1:1000, Cell Signaling Technology), rabbit anti-phospho-TBK1 (1:1000, Cell Signaling Technology), rabbit anti-phospho-IRF3 (1:1000, Cell Signaling Technology), rabbit anti-IFIT1 (1:1000, Cell Signaling Technology), human anti-SARS-CoV-2 Spike (CR3022, 1:5000), rabbit anti-SARS-CoV-2 nucleocapsid (Genetex, 1:1000), rabbit anti-NF-κB (1:500, Cell Signaling Technology), rabbit anti-phospho-NF-κB (1:1000, Cell Signaling Technology), mouse anti-Strep (1:1000, Biolegend; 1:1000, Genscript), rabbit anti-Actin (1:1000, Cell Signaling Technology), Streptavidin-HRP (1:1000, Jackson ImmunoResearch), species-specific HRP-Conjugated antibodies (1:10,000, Jackson ImmunoResearch). Alexa Fluor conjugated secondary antibodies (1:500, Life Technologies), and nuclear counterstain 4’,6’-Diamidino-2-phenylindole dihydrochloride- (DAPI; 1:1000, ACROS Organics).
4
Immunoblotting of Coronary Artery Proteins
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Protein extracts from the coronary artery were separated by SDS-PAGE (11–15% acrylamide). After being transferred to PVDF membranes and blocking for 2 h, primary antibody was added overnight, followed by washing in PBS, application of a secondary HRP-conjugated antibody and development using an ECL system (Absin Bioscience). Rabbit anti-HMGB1 (1:1,000; Abcam, ab18256), goat anti-RAGE (1:1,000; RD systems, AF1145), rabbit anti-NF-κB (1:1,000; Cell Signaling Technology, 8242) mouse anti-β-actin (1:1,000; Proteintech, 60,008-1-Ig) were used as primary antibodies. All western blots in this study were subjected to different exposures: From 10 s to 10 min, and the optimal exposures were selected for data presentation.
5
Multicolor Immunofluorescence Staining Protocol
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Paraffin-embedded tissue sections were deparaffinized, hydrated in a graded ethanol series, and quenched by antigen retrieval with a citrate buffer (10 mM Sodium citrate, 0.05% Tween 20, pH 6.0) at >95° C for 10 min. Tissue sections were blocked with 0.1% BSA for 20 min and incubated with the primary antibodies mouse-anti-cytokeratin-7 (cat. no. MA1-06316; Invitrogen), rabbit-anti-PTEN (cat. No. 9559S; Cell Signaling, Danvers, MA, USA), and rabbit-anti-NF-κB (cat. No. SC-109; Santa Cruz Biotechnology, Heidelberg, Germany) for 2 h at 37°C in a humid atmosphere followed by incubation with the secondary antibodies goat anti-mouseAF488 (cat. No. A11017; Invitrogen) or goat anti-rabbitAF647 (cat. No. A21246; Invitrogen). All antibodies diluted 1:200 were applied and incubated 1 h at 37°C under humidity. DAPI (1 µg/mL) (cat. No. D9542; Sigma Aldrich) was used for nuclei staining. Fluorescence was visualized and recorded using a Zeiss LSM 710 confocal laser scanning microscope (Carl Zeiss Microscopy GmbH).
6
Macrophage Epigenetic Regulation Study
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BMDMs were treated as indicated. Whole-cell lysates were prepared in RIPA buffer and subjected to western blotting. The antibodies used were rabbit anti-HP1-α, rabbit anti-H3K4me3, rabbit anti-H3K9me3, rabbit anti-H3K27me3, rabbit anti-H3K27Ac, rabbit anti-H, rabbit anti-NF-B, rabbit anti-ERK, rabbit anti-MAPK, rabbit anti-p-SAPK-JNK, rabbit anti-NF-κB, rabbit anti-ERK, rabbit anti-MAPK, rabbit anti-SAPK-JNK, mouse anti-actin, HRP-conjugated donkey anti-rabbit IgG and HRP-conjugated sheep anti-mouse IgG (all purchased from Cell Signaling Technology, Danvers, MA, USA). The signals were detected by chemiluminescence. The protein bands intensities were quantitated using ImageJ Gel Analysis program. The modified proteins were normalized to their loading controls (total forms) and relatively normalized to those of the unstimulated cells.
7
NF-κB and AKT Phosphorylation in IL-1β-Treated ACL Cells
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The NF-κB and AKT phosphorylation activity in interleukin (IL)-1β treatment of anterior cruciate ligament (ACL)-derived cells were measured. In brief, cells were treated with IL-1β in the absence or presence of 1 mM 4-MU, and cell lysate were collected and used for Western blotting analysis with the antibodies of rabbit anti-AKT (1:20, catalog no: 9272, lot22; Cell Signaling Technology), rabbit anti-phospho-AKT (1:20, catalog no: 9271, lot12; Cell Signaling Technology), rabbit anti-NF-κB (1:60, catalog no: 8242, lot16; Cell Signaling Technology), and rabbit anti- phospho-NF-κB (1:60, catalog no: 3033, lot17; Cell Signaling Technology).
8
Immunodetection of Alzheimer's Biomarkers
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Rabbit anti-APP (1:4000; Abcam: ab32136), rabbit anti-PS1 (1:1000; Abcam: ab134195), rabbit anti-NEP (1:1000; Abcam: ab126593), rabbit anti-IDE (1:1000; Abcam: ab133561), mouse anti-BACE1 (1:500; Abcam: ab183612), rabbit anti-BDNF (1:1000; Abcam: ab101747), rabbit anti-NGF (1:1000; Abcam:ab68151), rabbit anti-NF-κB (1:1000; Cell Signaling Technology: #8242), rabbit anti-Iba1 (1:1000; Abcam: ab178847), rabbit anti-CD40 (1:1000; Abcam: ab65853) rabbit anti-Synaptophysin (1:1000; Cell Signaling: #4329), rabbit anti-PSD-93 (1:1000; Cell Signaling: #9445), rabbit anti-PSD-95 (1:1000; Cell Signaling: #2507), and mouse anti-β-actin (1:60000; Sigma:A5441). Enzyme-linked immune sorbent assay (ELISA) kits: Mouse amyloid beta peptide 1-42 (15.6pg/mL /1000pg/mL) Emax immunoassay kit (CUSABIO: Lot: R09019069), Mouse TNF-α (6.25pg/mL /400pg/mL) Emax immunoassay kit (CUSABIO: Lot: Y05014334), Mouse IL-1β (62.5pg/mL /4000pg/mL) Emax immunoassay kit (CUSABIO: Lot: X20019335).
9
Protein Expression Analysis in Lung Tissue
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Proteins were extracted from lung tissues and cells. Target proteins were separated on 8%–12% sodium dodecyl sulphate‐polyacrylamide gels (according to the target protein sizes: 8% for collagen I, 10% for proteins in NF‐κB pathway, 12% for Cav‐1) and subsequently transferred onto polyvinylidene difluoride membranes. The membranes were blocked for 1 h in 5% bovine serum albumin (BSA) buffer and incubated with primary antibodies in 5% BSA at 4℃ overnight. The primary antibodies were as follows: rabbit anti‐caveolin‐1 (#3267, 1:5000), rabbit anti‐p‐NF‐κB(#3033, 1:1000), rabbit anti‐p‐IκBα (#2859, 1:1000), rabbit anti‐NF‐κB (#8242, 1:1000) and rabbit anti‐IκBα (#9242, 1:1000) from Cell Signaling Technology; rabbit anti‐collagen I (ab34710, 1:1000) from Abcam; mouse anti‐GAPDH (60004‐1‐Ig, 1:10,000), rabbit anti‐α‐tubulin (11224‐1‐AP, 1:5000) and mouse anti‐β‐actin (66009‐1‐Ig,1:20000) from ProteinTech. The membranes were incubated with horseradish peroxidase‐conjugated anti‐rabbit and anti‐rat for 60 min for detection. Protein expression was normalized using α‐tubulin and GAPDH or β‐actin.
10
Inflammatory Signaling Modulation Protocol
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Recombinant human epidermal growth factor (EGF), tumor necrosis factor-α (TNF-α), TGFβ1, and interleukin (IL)-1β were purchased from PeproTech (USA). Cigarette smoke condensate (CSC) was acquired from the Tobacco and Health Research Institute 26 (University of Kentucky, USA). Lipopolysaccharides (LPS), phorbol 12-myristate 13-acetate (PMA), Acrolein, and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich (USA). For the inhibition experiments, the IKK inhibitor (BAY 11-7082), PI3K inhibitor (LY294002), NF-κB inhibitor (Triptolide), and TGFβ receptor inhibitor (SB431542) were also purchased from Sigma-Aldrich. Rabbit anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH, sc-25778) was purchased from Santa Cruz Biotechnology (USA). Rabbit anti-phospho-Smad3 (#9520), mouse anti-HDAC2 (#5113), and rabbit anti-NF-κB (#8242) were obtained from Cell Signaling Technology (USA). Rabbit anti-acetyl-NF-κB at K310 (ab19870 for ChiP assay), mouse anti-MUC5AC (ab3649), and rabbit anti-Smad3 (ab28379) were acquired from Abcam (UK). Rabbit anti-acetyl-NF-κB at K310 (PA5-17264 for cell staining), Alexa Fluor 488-conjugated goat anti-rabbit IgG (A32731), and Alexa Fluor 568-conjugated goat anti-mouse (A11126) were obtained from Thermo Fisher Scientific (USA). The secondary antibodies for western analysis were acquired from GenDepot (USA).
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