Total protein was extracted from pancreatic, jejunal, and colonic tissues using membrane and cytoplasmic or nuclear and cytoplasmic protein extraction kits (Beyotime, Jiangsu, China) following the manufacturer's instructions. Equal amounts of protein for each sample were subjected to 5%–10% SDS-PAGE and then transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA). The membranes were blocked with 10% nonfat milk in Tris-buffered saline containing 0.1% (v/v) Tween-20 and incubated with primary antibodies against TLR4, MyD88, TNF receptor associated factor- (TRAF-) 6, IL-1β, TRIF, TRIF-related adaptor molecule (TRAM), IFN-β (Bioss, Beijing, China), nuclear factor- (NF-) κB p65 (Cell Signaling Technology), interferon regulatory factor- (IRF-) 3 (Santa, Dallas, TX), claudin-1, occludin, ZO-1 (Sangon, Shanghai, China), β-actin, histone H3 (Cell Signaling Technology), or Na/K ATPase (Proteintech, Rosemont, IL) at 4°C overnight. Subsequently, membranes were washed before incubation with the corresponding secondary antibodies (Zhongshan Goldenbridge, Beijing, China) for 2 h at 37°C. Finally, protein bands were visualized using an enhanced chemiluminescence detection kit (Beyotime). Na/K ATPase, β-actin, and histone H3 served as internal controls for membrane proteins, cytoplasmic proteins, and nucleoproteins, respectively.
Nuclear factor nf κb p65
Manufactured by Cell Signaling Technology
Sourced in China, United States
The Nuclear factor- (NF-) κB p65 is a protein that plays a central role in the regulation of gene expression related to immune response, inflammation, and cell survival. It is a subunit of the NF-κB transcription factor complex.
Automatically generated - may contain errors
Lab products found in correlation
Enhanced chemiluminescence detection kit, by Beyotime (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Ifn β, by Bioss Antibodies (1 mentions)
Na k atpase, by Proteintech (1 mentions)
Histone h3, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
E cadherin, by Proteintech (1 mentions)
N cadherin, by Proteintech (1 mentions)
Vimentin, by Proteintech (1 mentions)
Snail, by Cell Signaling Technology (1 mentions)
Phospho nf κb p65, by Cell Signaling Technology (1 mentions)
Bca protein assay kit, by Solarbio (1 mentions)
Gapdh, by Proteintech (1 mentions)
Ripa buffer, by Solarbio (1 mentions)
2 protocols using nuclear factor nf κb p65
1
Protein Extraction and Western Blot Analysis
2
Protein Isolation and Western Blot Analysis
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As described by Xie et al [14 (link)], all proteins were isolated by the RIPA buffer (Solarbio, Beijing, China) and the concentration of protein was tested using a BCA Protein Assay Kit (Solarbio). The proteins were subjected to 10% SDS-PAGE, followed by transfer of the separated proteins onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The main antibodies were ubiquitin-specific peptidase 2 (Proteintech, Rosemont, IL, USA, 1:1000), GAPDH (Proteintech, 1:5000), nuclear factor (NF)-κB p65 (Cell Signaling Technology, Danvers, MA, USA, 1:1000), phospho-NF-κB p65 (Cell Signaling Technology, 1:1000), N-cadherin (Proteintech, 1:5000), E-cadherin (Proteintech, 1:25,000), Vimentin (Proteintech, 1:5000), and Snail (Cell Signaling Technology, 1:1000). Secondary antibodies used were horseradish peroxidase-conjugated Affinipure Goat Anti-Rabbit IgG(H + L) and horseradish peroxidase-conjugated Affinipure Goat Anti-Mouse IgG(H + L) (Proteintech, 1:8000). Incubations with the antibodies were performed per the manufacturer’s instructions.
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