Mouse proteins were separated by SDS-PAGE and transferred to PVDF membrane. After blocking in TBS-T with 1% BSA in (TBS-T), the blots were incubated with primary antibody (1:1000 dilution) overnight. The rabbit monoclonal antibodies for LC3 (#12741), STAT1 (#9172), pSTAT1-Tyr701 (#9167), NFκB phospho-p65 (Ser536) (#3033), p62/SQSTM1 (#23214) and NFκB p65 (#8242) were obtained from Cell Signaling. The anti-NKLAM antibody has been described previously [6 (link)]. The blots were washed 3 times in TBS-T and incubated with horse radish peroxidase-conjugated secondary antibodies. The images were captured with a BioRad Chemidoc XRS+ imager. Protein normalization was performed using β-actin Western blot or with 2,2,2-trichloroethanol (TCE). Briefly, TCE (54 mM final concentration) was added to the polyacrylamide gel solution prior to polymerization. TCE binds to tryptophan residues and becomes fluorescent when exposed to UV light [14 (link)], allowing the visualization of the total protein in each lane. For normalization, the band intensity for a protein of interest was divided by the intensity of the TCE signal in the corresponding lane. For phosphoprotein analysis (pSTAT1 and pp65), the band intensity of the phosphorylated form of the protein was divided by the band intensity of the total protein.
Nfκb phospho p65 ser536
Manufactured by Cell Signaling Technology
Sourced in France
The NFκB phospho-p65 (Ser536) antibody is a laboratory research tool used to detect the phosphorylation of the p65 subunit of the NF-κB transcription factor at serine 536. This modification is involved in the regulation of NF-κB activity. The antibody can be used in various immunoassay techniques to analyze the phosphorylation status of p65 in cellular samples.
Automatically generated - may contain errors
Lab products found in correlation
Nf κb p65, by Cell Signaling Technology (2 mentions)
Pstat1 tyr701, by Cell Signaling Technology (1 mentions)
Sqstm1 p62, by Cell Signaling Technology (1 mentions)
Chemidoc xrs imager, by Bio-Rad (1 mentions)
Stat1, by Cell Signaling Technology (1 mentions)
Phospho sapk jnk thr183 tyr185, by Cell Signaling Technology (1 mentions)
Sapk jnk, by Cell Signaling Technology (1 mentions)
Supersignal west pico chemoluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Peroxidase conjugated secondary antibody, by Merck Group (1 mentions)
Las4000 apparatus, by Fujifilm (1 mentions)
Phospho erk1 2 thr202 tyr204, by Cell Signaling Technology (1 mentions)
Erk1 2, by Cell Signaling Technology (1 mentions)
β actin, by Merck Group (1 mentions)
2 protocols using nfκb phospho p65 ser536
1
Western Blot Analysis of Mouse Proteins
2
Western Blot Analysis of Cell Signaling
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Total protein extracts were performed as previously described [53] and quantified using bicinchoninic acid method following the manufacturer's instruction (Pierce). Western blot experiments were performed as previously described [52] on nitrocellulose membrane (0.2 µm, Schleicher et Shüll, Life Technologies).
Membranes were incubated with antibodies against MUC4 (8G7) (1/200, SC-53945 Santa Cruz), phospho-Erk1/2 (Thr202/Tyr204) (1/500, #4276, Cell Signaling), Erk1/2 (1/500, #4696, Cell Signaling), NF-κB Phospho-p65 (Ser536) (1/200, #3033, Cell Signaling), NF-κB p65 (1/500, #E498, Cell Signaling), phospho-SAPK/JNK (Thr183/Tyr185) (dilution 1/500, #9251, Cell Signaling), SAPK/JNK (1/500, #3258, Cell Signaling), β-actin (#A5441, 1/5000, Sigma-Aldrich , St Quentin Fallavier, France). Antibodies were diluted in 5 % (w/v) nonfat dry milk in 1X Tris-Buffered Saline containing Tween-20 (TBS-T), except for MUC4 and β actin that were diluted in 1X TBS-T, and incubated overnight at 4°C. Peroxidase-conjugated secondary antibodies (Sigma-Aldrich) were used and immunoreactive bands were visualized using the Super Signal® West Pico chemoluminescent substrate (Thermo Scientific) and visualized using the LAS4000 apparatus (Fujifilm, Courbevoie, France).
Membranes were incubated with antibodies against MUC4 (8G7) (1/200, SC-53945 Santa Cruz), phospho-Erk1/2 (Thr202/Tyr204) (1/500, #4276, Cell Signaling), Erk1/2 (1/500, #4696, Cell Signaling), NF-κB Phospho-p65 (Ser536) (1/200, #3033, Cell Signaling), NF-κB p65 (1/500, #E498, Cell Signaling), phospho-SAPK/JNK (Thr183/Tyr185) (dilution 1/500, #9251, Cell Signaling), SAPK/JNK (1/500, #3258, Cell Signaling), β-actin (#A5441, 1/5000, Sigma-Aldrich , St Quentin Fallavier, France). Antibodies were diluted in 5 % (w/v) nonfat dry milk in 1X Tris-Buffered Saline containing Tween-20 (TBS-T), except for MUC4 and β actin that were diluted in 1X TBS-T, and incubated overnight at 4°C. Peroxidase-conjugated secondary antibodies (Sigma-Aldrich) were used and immunoreactive bands were visualized using the Super Signal® West Pico chemoluminescent substrate (Thermo Scientific) and visualized using the LAS4000 apparatus (Fujifilm, Courbevoie, France).
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