Protein was extracted from mouse lung homogenates or cell lysates, then subjected to Western blot. The membranes were incubated with 1:1,000 dilution of purified rabbit anti-HMGB1 polyclonal antibody (ab79823, Abcam, USA), anti-NF-κB monoclonal antibody (8242, Cell signaling technology, USA), anti-phosphorylated NF-κB monoclonal antibody (3033, Cell signaling technology, USA) and anti-β-actin monoclonal antibody (Bioworld Technology, Inc.) overnight at 4°C. The immunoreactive bands were visualized using HRP-conjugated donkey anti-rabbit IgG (1 : 5000, 711-035-152, Jackson Immuno Research Laboratories, Inc.). The blots were quantified using BioRad Quantity One software 4.4.0 (Bio Rad Laboratories, Inc.).
Anti nf κb monoclonal antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The Anti-NF-κB monoclonal antibody is a laboratory tool used to detect and study the NF-κB protein, a critical regulator of cellular processes. It is a highly specific and well-characterized antibody that can be used in various experimental techniques, such as Western blotting, immunoprecipitation, and immunohistochemistry, to identify and quantify the NF-κB protein in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Hrp conjugated donkey anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Ab79823, by Abcam (1 mentions)
Anti β actin monoclonal antibody, by Merck Group (1 mentions)
Ap124p, by Merck Group (1 mentions)
Anti cleaved caspase 3 monoclonal antibody, by Cell Signaling Technology (1 mentions)
Anti parp polyclonal antibody, by Cell Signaling Technology (1 mentions)
Nanodrop 1000 spectrophotometry, by Thermo Fisher Scientific (1 mentions)
Edta free halt protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Anti histone h3 monoclonal antibody, by Cell Signaling Technology (1 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (1 mentions)
4 protocols using anti nf κb monoclonal antibody
1
Western Blot Analysis of HMGB1, NF-κB and β-Actin
2
Lung Tissue Preparation and Immunohistochemistry
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Lungs were prefixed in 4% paraformaldehyde for 1 hour, then fixed in 10% neutral formalin solution overnight, followed by paraffin embedding, and cut into 4 μm serial sections. Hematoxylin-eosin (H&E) staining was used for pathological observation. SP immunohistochemical staining was used to observe the expression and distribution of p120 and NF-κB, with anti-p120 monoclonal antibody (1 : 200, sc-1730, Santa Cruz) and anti-NF-κB monoclonal antibody (1 : 50, Cell Signal Technology).
3
Quantifying Protein Expression Changes
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A total of 3 × 105 cells were seeded in a 6-well plate and transfected with 5 μM SAE1 siRNA or nontarget sense RNA. All cell groups were lysed using the RIPA buffer. Subsequently, 50 μg protein of each sample was loaded into 10–12% SDS-PAGE wells and electrophoresis was conducted at 50 V for 4 h and then transferred from the gel to the PVDF membrane at 200 mA for 2 h. After 1 h incubation with a blocking buffer, the membranes were incubated with primary antibodies (anti-SAE1 polyclonal antibody (1:500; proteintech; 10229-1-AP), anti-cleaved caspase-3 monoclonal antibody (1:500; Cell signaling, MA, USA; #9664), anti-β-actin monoclonal antibody (1:20,000; Sigma; St Louis, MI, USA; A5541), anti-cyclin D1 monoclonal antibody (1:500; proteintech; 60186-1-lg), anti-PARP polyclonal antibody (1:500; Cell Signaling; #9542), anti-NF-κB monoclonal antibody (1:500; Cell Signaling; #6956), and anti-p-NF-κB monoclonal antibody (1:500; Cell Signaling; #3033)) overnight and with secondary antibodies (Goat anti-Rabbit (1:5000; Millipore, MA, USA; AP132P) and Goat anti-Mouse (1:5000; Millipore; AP124P)) for 90 min. Thereafter, an ECL substrate solution (Western Lightning, MA, USA; 205-14621) was used to detect specific bands with a Minichemi chemiluminescence imaging instrument (ThermoFisher Scientific Inc, MA, USA) (n = 4).
4
Nuclear Factor-κB Regulation by FAEW and Fluoxetine
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T98G cells were treated with FAEW and fluoxetine for 24, 48, and 72 h, MG-132 for 1 h, followed by TNF-α induction for 3 h. MG-132 was used as positive control drug. Nuclear protein extracts were prepared according to the NE-PER nuclear and cytoplasmic extraction reagent (Thermo Scientific, USA) supplemented with EDTA-free Halt Protease Inhibitor Cocktail (Thermo Scientific). Protein concentrations were measured with Nano drop 1000 spectrophotometry (Thermo Scientific). The densities of the protein bands were quantified by FluorChemQ software (Biozym Scientific Company, Oldendorf, Germany). Nuclear p65 levels were determined with an anti-NF-κB monoclonal antibody (1:3000, Cell signaling). Histone H3 protein levels served as the internal control, using the anti-Histone H3 monoclonal antibody (1:3000, Cell Signaling). The inhibition effects of FAEW and fluoxetine toward p65 protein expression were calculated by comparison with untreated TNF-α induction group after using the control group without TNF-α for the quantification of western blots.
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