The procedures of SDS-PAGE and immunoblotting were described as our previous studies (19 (link)). Briefly, to measure the expression of target molecules, proteins were acquired from cell lysates and quantified by BCA assay. Anti-mouse-IL-6 and anti-NFκB were from Abcam. Anti-A-FABP was from R&D system. Other antibodies, including anti-STAT3, anti-phospho-STAT3 (Tyr705), β-actin, GAPDH, were from Cell Signaling Technology.
Anti nf κb
Manufactured by Abcam
Sourced in United Kingdom, United States, China
Anti-NF-κB is a laboratory antibody product used for the detection and analysis of the NF-κB protein. NF-κB is a transcription factor that plays a crucial role in regulating the immune response and inflammatory processes. This antibody can be used in various immunoassay techniques to study the expression and activation of NF-κB in different biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin, by Abcam (9 mentions)
Anti p nf κb, by Abcam (8 mentions)
Anti gapdh, by Abcam (7 mentions)
Anti nrf2, by Abcam (6 mentions)
Pvdf membrane, by Merck Group (6 mentions)
Anti p38, by Abcam (5 mentions)
Anti p stat3, by Abcam (4 mentions)
Anti stat3, by Abcam (4 mentions)
Anti tlr4, by Abcam (4 mentions)
Anti ho 1, by Abcam (4 mentions)
Anti iκbα, by Abcam (4 mentions)
Anti erk1 2, by Abcam (4 mentions)
Anti stat3, by Cell Signaling Technology (3 mentions)
Anti myd88, by Abcam (3 mentions)
Anti caspase 1, by Abcam (3 mentions)
Anti bcl 2, by Abcam (3 mentions)
Ripa buffer, by Beyotime (3 mentions)
Anti bax, by Abcam (3 mentions)
Ripa lysis buffer, by Beyotime (3 mentions)
Anti tnf α, by Abcam (3 mentions)
71 protocols using anti nf κb
1
Protein Expression Quantification by SDS-PAGE and Immunoblotting
2
Western Blot Analysis of Apoptosis Markers
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Equivalent amounts of proteins lysates were separated by 10% SDS-PAGE, transferred to 0.22μm PVDF membranes (Millipore, MA, USA), Membranes were blotted blocked in 5% fat-free milk in Tris-buffered saline and Tween 20 (TBST) for 1 h at room temperature, followed by washing and incubated with anti-GATA3 (1:1000, Abcam), anti-Cleaved-PARP (1:1000, Cell Signaling Technology), anti-Cleaved-Caspase3 (1:1000, Abcam), STAT3(1:1000, Abcam), p-STAT3(1:1000, Abcam), anti-NF-κB (1:1000, Abcam) at 4°C overnight. Subsequently, the membranes were incubated with HRP-conjugated IgG for 2h at room temperature and detected with an enhanced chemiluminescence system. A GAPDH antibody was used as control. The experiment was performed in triplicate.
3
Western Blot Analysis of Lung Tissue
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The lung tissue of mice was collected and digested by pre-cooled tissue protein RIPA lysis buffer (Beyotime Institute of Biotechnology). The protein content in the sample was determined by a BCA kit after being denatured in boiling water. Proteins (~30 µg/lane) were separated by SDS-polyacrylamide gel electrophoresis on 10% gels, then transferred to PVDF membranes and blocked in 5% skim milk/TBS-0.1% Tween (TBST) solution for 1 h at room temperature. The membranes were then incubated with the following primary antibodies: Anti-TLR4 (1:1,000; cat. no. ab13556; Abcam), anti-MyD88 (1:1,000; cat. no. ab219413; Abcam), anti-NF-κB (1:1,000; cat. no. 8242; Cell Signaling Technology, Inc.) and anti-GAPDH (1:1,000; cat. no. ab9485; Abcam) overnight at 4°C. After washing with TBST three times, HRP-labeled secondary antibody (1:1,000; cat. no. ab7090; Abcam) was added to the membranes for 1 h at room temperature, then washed with PBS. ECL (GE Healthcare) was used to visualize the blots and images were captured using an ImageQuant gel imaging system (GE Healthcare Bio-Sciences). The optical density ratio of the target band was then calculated by ImageJ software (version 6.0; National Institutes of Health). GAPDH was used as the loading control.
4
Macrophage Response to CnpB Protein
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Bone marrow-derived macrophages (BMDMs) from female C57BL/6 mice were collected and cultured in RPMI 1640 supplemented with 15% fetal bovine serum (FBS), 100 U/mL Penicillin, 100 μg/mL Streptomycin, and 25% L929 culture supernatant. Murine alveolar macrophage MH-S and BMDM were seeded in 6-well plates at 1×106 cells/well in RPMI 1640 medium supplemented with 10% FBS and incubated overnight at 37°C with 5% CO2. Cells were then stimulated with different concentrations of endotoxin-removed CnpB protein for the time indicated in figure legends, and medium alone was used as control. Cells were collected at time points, and total RNA was extracted using Trizol and then quantified for qRT-PCR. For Western-blot analysis, cells were lysed by RIPA buffer (Solarbio, China) supplemented with protease inhibitor cocktail (Roche, Switzerland) and phosphatase inhibitor cocktail (EpiZyme, China) for total proteins extraction at indicated time points. Anti-LC3 antibody (Sigma, USA) and anti-NF-κB (Abcam, UK) were incubated as primary antibodies, and β-actin was used as the loading control.
5
Analysis of US28-Mediated Signaling Pathways
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THP-1 cells, transduced to express various constructs of HA-US28, were lysed in radioimmunoprecipitation assay (RIPA) buffer, and nuclei and cell debris were removed by centrifugation at 13,000 × g for 10 min at 4°C. Proteins were separated on 10% SDS-polyacrylamide gels and transferred to nitrocellulose membranes (Axygen; Corning). Incubations with primary and secondary antibodies were performed using 5% skimmed milk for 1 h each at room temperature. Proteins were detected using the following antibodies: anti-p42/p44 or phosphor-anti-p42/p44 antibodies or anti-MSK1 or anti-phosphor-MSK1 antibodies (serine 360) (all 1:1,000; Cell Signalling Technology, Danvers, MA) or anti-CREB or phosphor-CREB antibodies (S360) (both Merck). The secondary antibody used was chicken anti-rabbit horseradish peroxidase (Santa Cruz Biotech). Blots were developed with the use of enhanced chemiluminescence (GE Healthcare) and visualized with autoradiography film.
To detect cellular localization of NF-kB, cells were fractionated using REAP (r apid, e fficient, a nd p ractical) (93 (link)) and proteins detected using the following antibodies: anti-NF-κB (Abcam, Inc.), anti-p84 (Thermo), and anti-GAPDH (Millipore). Secondary antibodies used were chicken anti-rabbit and bovine anti-mouse horseradish peroxidase (both Santa Cruz Biotech).
To detect cellular localization of NF-kB, cells were fractionated using REAP (
6
Immunocytochemical Analysis of NF-κB Activation
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Immunocytochemistry for confocal microscopy was performed on NFκB. NHEKs were seeded in each well of the slide chamber at a density of 1×104 cells per well. The cells were treated with HDM (10 mg/ml) or C48/80 (10 µg/ml) for 6 hours. After being washed with phosphate buffered saline (PBS), cells were fixed in 4% paraformaldehyde in PBS for 10 minutes, and then permeabilized with PBS containing 0.5% Triton X-100 (PBS-T) for 15 minutes. The cells were incubated with PBS-T containing 1% bull serum albumin for 1 hour and stained with the anti-NFκB (1:150; Abcam, Cambridge, MA, USA) overnight at 4℃, followed by incubation with Alexa Fluor 488 goat anti-rabbit IgG (1:500; Invitrogen) for 1 hour. Images were visualized using confocal microscopy with a 20X objective on an LSM 510 laser scanning microscope (Carl Zeiss) and analyzed using the LSM 5 browser imaging software.
7
Immunofluorescence Analysis of Cellular Signaling
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After each treatment, the cells were washed with PBS, fixed in 4% paraformaldehyde for 30 min at room temperature, blocked with 5% (w/v) bovine serum albumin (BSA, Sigma Aldrich, USA) in PBST, and immunostained with anti-Nrf2 (1:100 dilution; Abcam, USA), anti-SIKE (1:100 dilution; Thermo Fisher Scientific, USA), anti-NF-κB (1:100 dilution; Abcam) and anti-p-TBK1 (1:100 dilution; Cell Signaling Technology, USA) antibody overnight at 4 °C, followed by incubation with a goat anti-mouse Alexa Fluor-488-conjugated secondary antibody and/or goat anti-rabbit Alexa Fluor-594-conjugated secondary antibody (Abcam). After washing with PBS, the cells were stained with DAPI (Beyotime, Shanghai, China) and observed under a fluorescence microscope. Immunofluorescence staining for liver sections was performed using anti-Nrf2 (1:100 dilution; Abcam) and anti-SIKE (1:100 dilution; Thermo Fisher Scientific) as described for cells with little modification. Immunofluorescence images were obtained using a fluorescence microscope. Images were analyzed with Image J software.
8
Western Blot Analysis of Protein Expression
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With the application of BCA protein assay kit (Pierce Chemical), the prepared protein samples with the aid of Protein extraction kit was quantified. Afterwards, 10% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis was adopted to divide and shift protein samples to polyvinylidene difluoride membranes (Sigma). Incubation in 5% skim milk for half an hour was to impede nonspecific binding. Subsequently, the membranes were probed with primary antibodies including anti‐HDAC4 (cat. no. ab235583; 1:1000; Abcam), anti‐MUC5AC (cat. no. ab198294; 1:20000; Abcam), anti‐SIRT1 (cat. no. ab189494; 1:1000; Abcam), anti‐phosphorylated (p)‐NF‐κB (cat. no. ab247871; 1:1000; Abcam), anti‐NF‐κB (cat. no. ab32360; 1:1000; Abcam), and anti‐GAPDH (cat. no. ab9485; 1:2500; Abcam) at 4°C overnight. The membranes were incubated with horseradish peroxidase (HRP)‐linked anti‐rabbit (cat. no. ab6721; 1:2000; Abcam) secondary antibody on the following day. The ECL detection system (Millipore, WBKLS0100) was utilized to visualize the protein bands and band intensity was determined on Image Lab™ Software (Bio‐Rad, Shanghai, China). GAPDH was used as a loading control.
9
Protein Expression Profiling by Western Blot
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Protein extracts were loaded onto and separated by SDS-PAGE resolution (Pulilai Co., Beijing, China), followed by transferred onto a PVDF membrane. Next, membrane was blocked and incubated with primary antibodies and HRP-conjugated secondary antibody. Primary antibodies used in current experiments were listed: anti-NF-κB (Abcam, ab32536), anti-phospho-NF-κB (S536) (Abcam, ab76302), anti-IκBα (Cell Signaling Technology, 4812), anti-phospho-IκBα (S32) (Cell Signaling Technology, 2859), anti-TRAF2 (Cell Signaling Technology, 4712), anti-STAT3 (Cell Signaling Technology, 12640), anti-phospho-STAT3 (Y705) (Cell Signaling Technology, 9145), anti-IFNGR2 (ABclonal Technology, A7558), and anti-PD-L1 (Proteintech, 66248-1-Ig). β-actin was applied as a control for protein loading. Gels were visualized using the ECL system (Amersham Biosciences, Uppsala, Sweden) to determine the expression level of targeted proteins.
10
Gastric Tumor Protein Expression Analysis
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Gastric tumor cells were lysed by 1% NP40 buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% SDS) containing protease inhibitor cocktail (Biyuntian, China). Protein quantification was performed using Protein Quantitative Analysis kit (Biyuntian, China). 20 µg protein was separated in 10% SDS gels and transferred to polyvinylidene fluoride Immobilon-membranes. Then protein samples were blocked with 5% Nonfat-Dried Milk and incubated with primary antibodies: anti-ITGB1 (1:1000, Abcam, UK), anti-SPP1 (1:1000, Abcam, UK), anti-YBX1 (1:1000, Abcam, UK), anti- NF-κB (1:1000, Abcam, UK) and anti-β-actin (1:1000, Abcam, UK). Samples were then incubated with HRP-conjugated secondary antibody (1:1000, Thermo fisher, USA) for 1 h at room temperature. The target proteins were visualized using the ECL detection kit (Thermo fisher, USA).
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