Bovine serum albumin (BSA) was obtained from Shanghai Hengyuan Technology Biology Co., Ltd (Shanghai, China). Glucose, Phenylmethylsulfonyl Fluoride (PMSF), and ethylene diamine tetraacetic acid disodium salt (EDTA) were obtained from Beijing solabo Technology Co., Ltd. (Beijing, China). Penicillin and streptomycin were bought from Sangon Biotech Inc. (Shanghai, China). Ethanol (purity > 99%) was obtained from Xilong Chemical Co., Ltd. (Guangdong, China). Fetal bovine serum (FBS) was bought from Sangon Biotech, Inc. (Shanghai, China) and Dulbecco’s modified eagle medium (DMEM) was purchased from ThermoFisher (MA, USA). CCK-8 kit was obtained from Shanghai Yanjin Biotechnology Co., Ltd. (Shanghai, China). Anti-p-P38, anti-P38, anti-JNK, anti-p-JNK, anti-NF-κB p65, anti-p-NF-κB p65, anti-IκB, anti-p-IκB, anti-AKT and anti-p-AKT antibodies were purchased from Abcam Technology (Cambridge, UK). Anti β-actin antibody, Goat anti-rabbit and mouse IgG and mouse anti-goat secondary antibodies were purchased from ZSGB Biotech Co., Ltd. (Beijing, China). Unless specified, all other reagents are obtained from Sigma Chemical Co. (St. Louis, MO, USA).
Anti p nf κb p65
Manufactured by Abcam
Sourced in United States, United Kingdom
Anti-p-NF-κB p65 is a primary antibody that recognizes the phosphorylated form of the NF-κB p65 subunit. NF-κB p65 is a transcription factor that plays a key role in regulating the immune response and inflammation.
Automatically generated - may contain errors
Lab products found in correlation
Anti nf κb p65, by Abcam (4 mentions)
Anti β actin, by Abcam (4 mentions)
Ethanol, by Xilong (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Anti p38, by Abcam (1 mentions)
Anti p p38, by Abcam (1 mentions)
Anti jnk, by Abcam (1 mentions)
Anti p jnk, by Abcam (1 mentions)
Anti iκb, by Abcam (1 mentions)
Dulbecco s modified eagle medium dmem, by Thermo Fisher Scientific (1 mentions)
Anti p iκb, by Abcam (1 mentions)
Penicillin, by Sangon (1 mentions)
Anti akt, by Abcam (1 mentions)
Streptomycin, by Sangon (1 mentions)
Fetal bovine serum (fbs), by Sangon (1 mentions)
Anti notch1, by Abcam (1 mentions)
Anti hes1, by Abcam (1 mentions)
Anti collagen 2, by Abcam (1 mentions)
Anti p erk, by Abcam (1 mentions)
8 protocols using anti p nf κb p65
1
Reagents and Antibodies for Cell Assays
2
Chondrocyte Protein Extraction and Western Blot
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The reagent RIPA Lysis Buffer (Thermo Fisher Scientific) was used to extract the total protein of the cultivated chondrocyte or isolated cartilage tissue. RIPA was supplemented with protease inhibitor PMSF. Protein concentration was determined by a BCA kit (Thermo Fisher Scientific). Protein was mixed with protein loading buffer (Thermo Fisher Scientific) and loaded to a 12% SDS-PAGE gel for electrophoresis. Afterward, the gel was transferred to PVDF membrane (Thermo Fisher Scientific). After a blocking step with a blocking buffer, the membrane was incubated with primary monoclonal antibodies at 4 °C overnight. After washing, the membrane was incubated with secondary antibody. Finally, signal was detected by an ECL method. The primary antibodies used in this study includes anti-Aggrecan (1:1000, Abcam), anti-Collagen II (1:1000, Abcam), anti-β-actin (1:3000, Abcam), anti-ADAM8 (1:1000, Abcam), anti-p-ERK (1:1000, Abcam), anti-ERK (1:2000, Abcam), anti-p- NF-κB p65 (1:1000, Abcam), anti-NF-κB p65 (1:2000, Abcam), anti-MMP9 (1:1000, Abcam), anti-Notch1 (1:1000, Abcam), anti-Hes1 (1:2000, Abcam). All the primary antibodies were incubated for 4 h at 37 °C. The HRP-conjugated secondary antibody (1:5000, Sigma-Aldrich) was incubated for 2 h at RT.
3
Analyzing Signaling Pathways in Tumor Tissue
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The activities of CASP3, NF-κB(p65), and glycogen synthase kinase 3 beta (GSK-3β) were detected in tumor tissue and HeLa cells in vivo and in vitro from mice and cells treated with FSH. The nuclear or cytoplasmic fraction was extracted from every sample using the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions. The total protein levels of all samples were assayed with a BCA Protein Assay Kit using monoclonal antibodies to anti-β-actin, anti-CASP3, anti-pNF-κB(p65), and anti-pGSK-3β (all from Abcam, Cambridge, UK).
4
Proteomic Analysis of BMSC Proteins
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Proteins were extracted from more than 106 harvested BMSCs according to the kit protocol (KeyGen Biotech, China). [46 (link)] BMSCs were digested in a lysis buffer cocktail and centrifuged at 12000 g for 15 minutes at 4 °C to get the supernatant as the total protein. 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) was used for electrophoresis of protein (20 μg) with loading buffer and then blotted to a polyvinylidene fluoride (PVDF) membrane (Millipore Co., USA). Membranes were blocked with 5% skimmed milk in Tris-buffered saline tween 20, TBST, and then incubated overnight at 4°C with primary antibodies; anti-IHH, anti-P53, anti-P16, anti-PI3K, anti-p-PI3K, anti-Akt 1, anti-p-Akt 1, anti-NF-κB, anti-p-NF-κB-p65, anti-STAT3, anti-p-STAT3, anti-4EBP1, anti-p- 4EBP1, anti-p70S6K, anti-p-p70S6K and anti-β-actin (1:1000), all from Abcam, USA. The membranes were incubated for 1hour at RT with secondary antibody conjugated with horseradish peroxidase. Finally, TBST-washed membranes were treated with enhanced chemiluminescent (ECL) for detection (Bio-Rad, USA). Image Lab detection system (Bio-Rad, USA) was used for protein bands imaging and analysis.
5
Western Blot Analysis of Nasal Mucosal Proteins
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Protein levels were determined by western blotting. To extract total protein from mice nasal mucosal tissues, we employed RIPA lysate (Beyotime, Shanghai, P0013B) and PMSF (Beyotime, Shanghai, ST506). Before denaturation, protein concentration was determined using a BCA protein concentration determination kit (Beyotime, Shanghai, P0010S). To prepare a 10% gel for electrophoresis, an SDS-PAGE gel kit (Beyotime, Shanghai, P0012AC) was utilized. After blocking in 5% nonfat milk, antibodies were incubated: Anti-IL-1β (Abcam, 1 : 1000, ab283818), Anti-COX-2 (Proteintech, 1 : 3000, 66351-1-Ig), Anti-p-NF-κB p65 (Abcam, 1 : 1000, ab239882), Anti-NF-κB p65 (Abcam, 1 : 1000, ab16502), and Anti-β-actin (Abcam, 1 : 1000, ab16502) (Proteintech, 1 : 5000, 66009-1-Ig). Finally, strip development is carried out. ImageJ was used to examine the band density.
6
Protein Expression Analysis in Atrial Tissue
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Western blot analysis was performed as previously described.25 Proteins were extracted from atrial tissue and resolved by SDS‐PAGE (10% gels) and transferred to polyvinylidene difluoride (Millipore, Hong Kong, China) membranes. After blocking, the membranes were incubated with the following the primary antibodies: anti‐α‐SMA rabbit antibody (1:500; Abcam), anti‐transforming growth factor β1 rabbit antibody (1:1000; Abcam), anti‐TLR4 rabbit antibody (1:1000; Affbiotech, Cincinnati, OH), anti‐p‐NF‐κB p65 (1:500; Abcam), anti‐NF‐κB p65 rabbit antibody (1:3000; Abcam), anti‐p‐Smad2/3 (1:100; Abcam), anti‐Smad2/3 (1:100; Abcam) rabbit antibody, and anti‐GAPDH rabbit antibody (1:10000; Abcam). After washing, membranes were incubated with horseradish peroxidase‐goat anti‐rabbit‐conjugated secondary antibodies (1:10000; ASPEN, Massachusetts, USA), and protein bands were visualized by enhanced chemiluminescence (ASPEN). The protein expression levels were normalized to GAPDH protein.
7
Comprehensive Protein Extraction and Detection
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Total proteins were extracted from tissues or cells using RIPA lysis buffer (containing 1% PMSF) and then denatured at 100 °C using a Heating Block (Hangzhou Bioer Technology, China). Equal amounts of proteins were separated by SDS-PAGE and then transferred onto PVDF membrane. After blocking with 5% skim milk and incubation with primary antibody and secondary antibody, the immunoreactive bands on the membrane were reacted with ECL solution and detected by the Chemidoc EQ system (BioRad, USA). Primary antibodies used in this study were as follows: anti-GAPDH (1:5000, Bioworld, China), anti-CCND1 (1:2000, Bioworld, China), anti-CDK4 (1:2000, Bioworld, China), anti-CDKN1A (1:500, Bioworld, China), anti-CDKN1B (1:1000, Bioworld, China), anti-NF-κB p65 (1:2000, Abcam, USA), anti-p-NF-κB p65 (1:2000, Abcam, USA), anti-Galectin-1 (1:500, Santa Cruz, USA), anti-Ubiquitin (1:200, Santa Cruz, USA), anti-Smurf1 (1:500, Santa Cruz, USA), anti-CUL4A (1:1000, Abnova, China). For each experiment, the blots were from the same experiment and were processed in parallel.
8
TRAIL Ligand and Death Receptor Analysis
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Recombinant human TRAIL/Apo2 Ligand was purchased from Peprotech (Rocky Hill, NJ, USA). PE-conjugated human CD133/1 antibody was purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). PE-conjugated human DR5 and DR4 antibodies were purchased from BioLegend (San Diego, CA, USA). The following antibodies were used in western blot: anti-NF-κB/p65 1:1000 (Cell Signaling Technology (CST), Danvers, MA, USA), anti-p-NF-κB/p65 1:1000 (CST), anti-IκBα 1:1000 (CST), anti-p-IκBα 1:1000 (CST), anti-cFLIP (cellular FLICE-like inhibitory protein) 1:1000 (Abcam ab167409, Cambridge, MA, USA), anti-caspase-8 1:1000 (CST), anti-caspase-3 1:1000 (CST), anti-DR5 1:1000 (CST), anti-TNFRSF10A (DR4) 1:500 (ABGENT AP13702b, San Diego, CA, USA), anti-GAPDH 1:500 (Boster, Wuhan, China) and anti-β-actin 1:500 (Boster, Wuhan, China).
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