Phosphorylation of NF-κB p65 at serine 536 in pCECs after activation was investigated by flow cytometry as previously described 42 (link). Briefly, pCECs were harvested 5 minutes after stimulation with hTNF-α, fixed, permeabilized, and incubated successively with anti-phospho NF-κB p65 antibody (Cell Signaling, Danvers, MA), followed by staining with phycoerythrin-conjugated anti-rabbit IgG antibody (Cell Signaling). Rabbit IgG antibody (isotype) (Cell Signaling) was used as a negative control.
Anti phospho nf κb p65 antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The Anti-phospho-NF-κB p65 antibody is a laboratory reagent used to detect the phosphorylated form of the NF-κB p65 subunit. NF-κB is a transcription factor that plays a crucial role in regulating various cellular processes, including immune response, inflammation, and cell survival. The antibody can be used in techniques such as Western blotting, immunohistochemistry, and immunocytochemistry to study the activation and regulation of the NF-κB signaling pathway.
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Lab products found in correlation
Anti nf κb p65 antibody, by Cell Signaling Technology (3 mentions)
Lsab kit, by Agilent Technologies (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Phycoerythrin conjugated anti rabbit igg antibody, by Cell Signaling Technology (1 mentions)
Rabbit igg antibody isotype, by Cell Signaling Technology (1 mentions)
Facscelesta, by BD (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Chemiluminescence, by Santa Cruz Biotechnology (1 mentions)
Anti nf κb p65, by Santa Cruz Biotechnology (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
Anti cleaved caspase 3 antibody, by Abcam (1 mentions)
Anti mettl3 antibody, by Abcam (1 mentions)
Anti mmp2 antibody, by Proteintech (1 mentions)
Anti fibronectin antibody, by Abcam (1 mentions)
Anti mettl14 antibody, by Cell Signaling Technology (1 mentions)
Anti wt 1 antibody, by Abcam (1 mentions)
Anti podocin antibody, by Abcam (1 mentions)
Anti igf2bp2 antibody, by Proteintech (1 mentions)
Anti wtap antibody, by Proteintech (1 mentions)
Anti nephrin antibody, by Abcam (1 mentions)
11 protocols using anti phospho nf κb p65 antibody
1
Phosphorylation of NF-κB p65 in pCECs
2
Identification and Analysis of Mouse Neutrophils
3
Western Blot Analysis of NF-κB Signaling
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Cells were lysed in Lysis buffer (1% Nonidet P-40, 50 mmol/L Tris, pH 8.0, 50 mM NaCl, 1 mM EDTA, 10% glycerol with protease and phosphatase inhibitors) and followed by protein extraction. The amount of proteins was quantified with Bradford assay (Biorad). The cell lysates were loaded into polyacrylamide denaturing gels (12%) and transferred to polyvinylidene difluoride (PVDF) membranes (Merck Millipore, Kenilworth, NJ, United States). These membranes were then blocked in 5% milk with 0.1% Tween 20-PBS (PBS-T) solution for one hour. These primary antibodies were used: anti-NFκB-p65 and anti-β-actin antibody (HRP-conjugated) from Santa Cruz Biotechnology (Santa Cruz, CA, United States), anti-phosphoNFκB-P65 antibody from Cell Signaling Technologies, Danvers, MA, United States. Respective second antibodies were applied and washed before exposure for Chemiluminescence (Santa Cruz Biotechnology).
4
Investigating the Role of m6A Modification in Diabetic Nephropathy
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The following reagents were used: transfection was performed with Lipofectamine RNAiMAX Reagent (Invitrogen Life Technologies, 13, 778-030) according to the manufacturer’s protocol. The following antibodies were used: anti-β-actin antibody (Proteintech, 66009-1-Ig); anti-m6A antibody (Synaptic Systems, 202,003); anti-METTL3 antibody (Abcam, Ab195352, ZEN-BIOSCIENCE [382,974]); anti-METTL14 antibody (Cell Signaling Technology, 51, 104); anti-WTAP antibody (Proteintech, 10200-1-AP); anti-KIAA1429 antibody (SAB, 29, 774); anti-TIMP2 antibody (Abcam, ab180630); anti-NF-κB p65 antibody (Cell Signaling Technology, 8242); anti-phospho-NF-κB p65 antibody (Cell Signaling Technology, 3033); anti-cleaved caspase3 antibody (Abcam, Ab2302); anti-NOTCH3 antibody (Proteintech, 55114-1-AP); anti-NOTCH4 antibody (Affinity, DF13597); anti-WT-1 antibody (Abcam, ab267377); anti-nephrin antibody (Abcam, ab216341); anti-podocin antibody (Abcam, ab181143); anti-MMP2 antibody (Proteintech, 10373-2-AP); anti-MT1-MMP antibody (Proteintech, 14552-1-AP); anti-IGF2BP2 antibody (Proteintech, 11601-1-AP); anti-col-Ⅳ antibody (Abcam, ab6586); and anti-fibronectin antibody (Abcam, ab6586). STZ was purchased from Sigma Chemical Company (MO, USA). The PAS kits were obtained from Solarbio (Beijing, China). Mouse Albumin ELISA Kit was obtained from Abcam Biotechnology (Cambridge, UK).
5
NF-κB Pathway Protein Detection
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Proteins were isolated by RIPA buffer containing phosphatase and protease inhibitors (Roche, US). Equal total proteins were separated by SDS/PAGE gels and blotted onto PVDF membranes (Millipore, USA), followed by 5% milk blocking. The membranes were incubated overnight at 4°C with anti-phospho-NF-κB p65 antibody (#3033; Cell Signaling Technology), anti-NF-κB p65 antibody (#8242; Cell Signaling Technology), anti-PFKFB3 antibody (#13123S; Cell Signaling Technology), or anti-GAPDH antibody (60004-1-Ig; Proteintech). Finally, the blot was observed via the ECL detection system.
6
Curcumol Modulates NF-κB Pathway
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Curcumol, curcumin (Chengdu Manste Biotechnology Co., Ltd.), DMEM high sugar culture liquid [Thermo Fisher biochemical products (Beijing) Co., Ltd.], fetal bovine serum (FBS) (Biological Industries), CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS), Red Tashan cigarette (Hongta Tobacco Co., Ltd.), SYBR Premix EX Tap II fluorescent quantitative reagent box (Takara Biological Engineering Co., Ltd.), Anti-Phospho-NF-κB p65 antibody (Cell Signaling Technology Company), Anti-Smad2+Smad3 (phosghoT8) antibody (Abcam Company), NF-κB inhibitor Bay 11-7082 (Beyotime Biotech Company), DCFH-DA (Sigma Company), and BCA protein concentration assay kit (Beyotime Biotech Company) were used in the study.
7
Histological Analysis of Colorectal Tissues
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The livers, kidneys, white adipose tissues, and colorectal tissues were excised. The colon and rectum were opened longitudinally and fixed on a filter paper in 10% buffered formalin for more than 24 h. It was then divided into a rectal portion (approximately 1 cm in length on the oral side from the dentate line) and a colonic portion. Each segment and the other tissues were paraffin embedded. From the rectal tissue blocks, two serial sections were subjected to hematoxylin and eosin (HE) staining for histopathology to evaluate the development of BCACs [16 (link)]. Immunohistochemical staining for phospho-nuclear factor-κB (NF-κB) p65 was run on histological sections to estimate NF-κB activity, respectively, in the colonic crypts [11 (link),26 (link)], using the LSAB Kit (DAKO, Glostrup, Denmark) with primary antibody, anti-phospho-NFκB p65 antibody (a final dilution of 1:50, Ser276; Cell Signaling Technology, Danvers, MA, USA). The positive cell index (%) for phospho-NF-κB p65 was determined based on previous methods [15 (link),47 (link)].
8
Quantifying Colonic Crypt Biomarkers
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Immunohistochemistry for β-catenin was performed using the labeled streptavidin-biotin method (LSAB Kit; DAKO, Glostrup, Denmark) to count the number of BCAC [12 (link),23 (link),24 (link)]. The primary antibody for β-catenin (BD Transduction Laboratories, San Jose, CA, USA) was used at a final dilution of 1:1000. Immunohistochemical staining for proliferating cell nuclear antigen (PCNA), which is a G1-to-S phase marker, and for phospho-nuclear factor-κB (NF-κB) p65 were run on histological sections to estimate cell proliferative activity and NF-κB activity, respectively, in the colonic crypts [11 (link),23 (link)], using the LSAB Kit (DAKO) with primary antibodies, anti-PCNA antibody (a final dilution of 1:100, Santa Cruz Biotechnology, Dallas, TX, USA) and anti-phospho-NF-κB p65 antibody (a final dilution of 1:50, Ser276; Cell Signaling Technology, Danvers, MA, USA). The PCNA-labeling index (%) and positive cell index (%) for phospho-NF-κB p65 were determined based on previous methods [11 (link),23 (link)].
9
Analysis of Cartilage Protein Expression
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Anti-Collagen II antibody (ab34712, Abcam), antiphospho-NF-κB p65 antibody (3033, Cell Signaling Technology), anti-GAPDH (G8795, Sigma-Aldrich) and anti-G1 aggrecan antibody (generated by the late Dr Peter Roughley) (Roughley and Mort, 2012) were used. Anti-rabbit secondary antibodies were purchased from Jackson ImmunoResearch (111-035-144, West Grove, PA, USA).
10
Protein Expression Analysis via Western Blot
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Protein Extraction Kit was employed to isolate the protein according to the manufacturer’s instructions. Following separation using sodium dodecyl sulfate–polyacrylamide gel electrophoresis, the samples were transferred to a 0.22-μm PVDF membrane. The membranes were incubated with the primary antibody at 4 degree celsius overnight. Primary antibodies targeting IL-1β (#26,048–1-AP, 1:500), TNF-α (#60,291–1-AP, 1:500), and NF-κB P65 (#10,745–1-AP, 1:800) were obtained from by Proteintech (Wuhan, China), whereas anti-phospho-NF-κB p65 antibody (#3033, 1:500) was derived from Cell Signaling Technology. The PVDF membranes and horseradish peroxidase–conjugated secondary antibodies were incubated for 50 min with gentle shaking. The protein bands were visualized by an ECL kit (#GPP1824; GenePool Biotechnology, Beijing, China). Finally, densitometry values of the bands were obtained using Quantity One version 4.6.2.
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