The cells were washed with PBS before being lysed in lysis buffer that can extract both cytoplasm proteins and nucleoproteins. The sample was stored at −80°C. Samples containing an equal amount of proteins were mixed with 5x SDS loading buffer and electrophoresed on a 10% SDS-PAGE gel. The proteins were then transferred onto PVDF membrane (Millipore). The membranes were blocked and probed with antibodies anti-NFAT2 (CST), TRAF6 (Immunoway), p-NF-κBp65 (Immunoway), PCNA (Immunoway), p65 (Immunoway), and β-actin (Proteintech group). The membranes were incubated with specific primary antibodies overnight at 4°C at a 1 : 1000 dilution. Subsequently, the membranes were washed with TBS/T for 15 min, three times, and incubated for 1 h with HRP-conjugated secondary antibodies (Abbkine). The protein was visualized with chemiluminescence, and a densitometric scanner was used to determine the density of the band. All experiments were repeated at least three times independently.
P nf κbp65
Manufactured by Immunoway
Sourced in China
The P-NF-κBp65 is a lab equipment product that measures the phosphorylation of the NF-κBp65 protein. NF-κBp65 is a subunit of the NF-κB transcription factor complex, which plays a crucial role in various cellular processes, including immune response, inflammation, and cell survival. The P-NF-κBp65 assay allows for the quantitative assessment of NF-κBp65 phosphorylation, which is an important indicator of NF-κB activation.
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Lab products found in correlation
Hrp conjugated secondary antibody, by Abbkine (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
β actin, by Proteintech (1 mentions)
Traf6, by Immunoway (1 mentions)
Proliferating cell nuclear antigen (pcna), by Immunoway (1 mentions)
Bca protein quanti cation kit, by Vazyme (1 mentions)
P yap1, by Cell Signaling Technology (1 mentions)
Cleaved caspase 1, by Immunoway (1 mentions)
Tnf α, by Cell Signaling Technology (1 mentions)
Cleaved caspase 7, by Cell Signaling Technology (1 mentions)
Il 1β, by Bioss Antibodies (1 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Nf κb, by Cell Signaling Technology (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Nlrp3, by Proteintech (1 mentions)
β actin, by Immunoway (1 mentions)
Il 18, by ABclonal (1 mentions)
Gsdmd, by ABclonal (1 mentions)
2 protocols using p nf κbp65
1
Western Blot Analysis of Cellular Proteins
2
Evaluating Apoptosis and Inflammation Markers in Cell Lysates
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All cells were lysed with RIPA lysis buffer (Beyotime, Shanghai, China) containing protease and phosphatases inhibitors (1μg/ml).Theprotein concentrationwas determined using BCA Protein Quanti cation Kit (Vazyme, Nanjing, China).Denaturedproteins were separated via 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes (0.45μm).The membrane was incubated with the corresponding antibodies againstLATS1 (1:1,000, Cell Signaling Technology),Yap1 (1:1,000, Cell Signaling Technology), P-YAP1 (1:1000, Cell Signaling Technology) Bcl2(1:1000,Cell Signaling Technology),Bax(1:1000,Cell Signaling Technology),Cleaved-caspase3(1:1000, Cell Signaling Technology),Cleaved-caspase7(1:1000, Cell Signaling),Cleaved-caspase-1(1:1000, Immunoway),TNF-α(1:1000, Cell Signaling),IL-1β (1:1000, Bioss),IL-18(1:1000, ABclonal),P-NF-κB/P65(1:2000,Immunoway),NF-κB (1:1000, Cell Signaling Technology),GSDMD ( 1:1000,Abclonal),NLRP3 (1;1000,proteintech) and NLRC4 (1:1000,ABclonal).β-actin (1:10000, Immunoway) was used as an endogenous reference.
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