PTNs were a generous gift from Won-Kyo Jung, Pukyong National University, Busan, South Korea. PTNs were prepared from Ecklonia cava collected along the Jeju Island coast of Korea as previously described [43 (link)]. Composition and chemical structures of PTNs in the ethanolic extracts were previously characterized [44 (link),45 (link)]. EasyefTM, a commercial spray-type ointment containing rhEGF, was purchased from Daewong Pharmaceuticals (Seoul, South Korea). The primary antibodies used were as follows: anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (#5174), anti-p65-NF-κB (#8242), anti-IL-1β (#12242), and anti-HO-1(heme oxygenase) (#5853) purchased from Cell Signaling Technology (Danvers, MA, USA); anti-ASC (SC514414) and anti-NRF2 (SC1722) purchased from Santa Cruz Biotechnology (Dallas, TX, USA); anti-AQP3 (AB3276) purchased from Merck Millipore (Burlington, MA, USA); and anti-COX2 (#610203) purchased from BD Biosciences (Franklin Lakes, NJ, USA). Horseradish peroxidase (HRP)-conjugated secondary anti-rabbit and anti-mouse antibodies were purchased from Cell Signaling Technology.
Anti nf κb p65
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom, China, Germany, Japan
Anti-NF-κB p65 is a primary antibody that recognizes the p65 subunit of the NF-κB transcription factor. NF-κB is a key regulator of gene expression involved in various cellular processes, including immune response, inflammation, and cell survival.
Automatically generated - may contain errors
Lab products found in correlation
Anti phospho nf κb p65, by Cell Signaling Technology (52 mentions)
Anti iκbα, by Cell Signaling Technology (45 mentions)
Anti p nf κb p65, by Cell Signaling Technology (31 mentions)
Anti akt, by Cell Signaling Technology (24 mentions)
Anti β actin, by Cell Signaling Technology (24 mentions)
Anti gapdh, by Cell Signaling Technology (24 mentions)
Pvdf membrane, by Merck Group (21 mentions)
Anti β actin, by Merck Group (20 mentions)
Anti β actin, by Santa Cruz Biotechnology (16 mentions)
Anti phospho iκbα, by Cell Signaling Technology (15 mentions)
Anti phospho nf κb p65 ser536, by Cell Signaling Technology (15 mentions)
Anti p iκbα, by Cell Signaling Technology (14 mentions)
Anti iκb, by Cell Signaling Technology (14 mentions)
Anti stat3, by Cell Signaling Technology (14 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (14 mentions)
Anti erk1 2, by Cell Signaling Technology (13 mentions)
Anti phospho akt, by Cell Signaling Technology (13 mentions)
Anti p38, by Cell Signaling Technology (13 mentions)
Anti p38 mapk, by Cell Signaling Technology (12 mentions)
Anti jnk, by Cell Signaling Technology (12 mentions)
293 protocols using anti nf κb p65
1
Ecklonia cava-Derived Polyphenol Compounds
2
Western Blot Analysis of Protein Markers
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A total of 25 μg protein from each sample was separated on 10% Bis-Tris polyacrylamide gel through electrophoresis and then blotted onto PVDF (polyvinylidene fluoride) membranes (Millipore). Blots were immunostained with primary antibodies, including rabbit anti-EZH2 (Cell Signaling Technology), anti-VOPP1 (Santa Cruz), anti-Ki-67 (Santa Cruz), anti-p65-NF-κB (Cell Signaling Technology), anti-E2F-1 (Cell Signaling Technology), anti-pAKT (Phospho-Thr308; Cell Signaling Technology), anti-pERK (Phospho-Thr980; Cell Signaling Technology), anti-TS (Abcam), and anti-β-actin (Cell Signaling Technology) at 4°C overnight and with secondary antibody at room temperature for 1 hr. Immunoblots were visualized using Immobilon Western Chemiluminescent HRP Substrate (Millipore). β-actin was used as the internal control.
3
Western Blot Analysis of ATB0,+ Expression
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For the determination of ATB0,+ expression, cells were lysed in a RIPA buffer added with a cocktail of protease inhibitors (Complete, Mini, EDTA-free, Roche). The Western Blot analysis was performed as described in a previous study (28 (link)). Briefly, 20 μg of proteins were separated on the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (4–12% acrylamide) and were electrophoretically transferred to polyvinylidene fluoride (PVDF) membranes (Immobilione-P membrane, Merck, NJ, USA). Membranes were first incubated for 1 h at room temperature (RT) in a Tris-buffered saline solution (TBS; 50 mM Tris-HCl pH 7.5, 150 mM NaCl) containing 5% non-fat dried milk and then incubated overnight at 4°C in a Tris-buffered saline with Tween 20 (TBST) buffer added with 5% bovine serum albumin (BSA) and anti-ATB0,+ purified rabbit polyclonal antibody (1:5,000, Merck, NJ, USA), anti-p65 NF-κB, or anti-phospho-p65 (Ser536) NF-κB (1:2000, Cell Signaling TECHNOLOGY, MA, USA). Vinculin, detected with a monoclonal antibody (1:2,000; Merck, NJ, USA), was employed as internal standard. Immunoreactivity was visualized by using the Immobilon Western Chemiluminescent HRP Substrate (Merck, NJ, UA). Western Blot images were captured by using an iBright FL1500 Imaging System (ThermoFisher Scientific, MA, USA) and analyzed with the iBright Analysis Software.
4
Molecular Mechanisms in Inflammatory Response
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Angiotensin II and JSH-23 were purchased from Sigma-Aldrich (St. Louis, MO). The primary antibodies were obtained from the following sources: anti-LC3, anti-p-p65 NF-κB (Ser536), and anti-p65 NF-κB (Cell Signaling Technology, Beverly, MA), anti-ATG5, anti-IL-1β, anti-F4/80, anti-CD3, and anti-p-H3 (Abcam, Cambridge, MA); Anti-p62 GeneTex (Irvine, CA); anti-FLAG and anti-HA (Invitrogen Life Technologies, Paisley, UK); Alexa Fluor 488 goat anti-rabbit IgG antibody, Alexa Fluor 488 goat anti-mouse IgG antibody, Alexa Fluor 546 goat anti-mouse IgG antibody, and Alexa Fluor546 goat anti-rabbit IgG antibody (Invitrogen Life Technologies, Paisley, UK).
5
Quantitative Western Blot Analysis of NF-κB Pathway
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Cellular extracts from THP-1 were prepared according to standard protocol [54 (link)]. Total protein concentration was determined using the colorimetric reagent of the Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA). To determine specific protein contents, cytoplasmic and nuclear extracts (50 μg each) were separated in a 4–20% Ready Gel Tris-HCl gel (Bio-Rad Laboratories) and then transferred to a nitrocellulose membrane. The membranes were blocked in TTBS with 5% milk for 1 h at room temperature and incubated with primary antibodies, including anti-p65-NF-κB and IκB (both from Cell Signaling Technology, Danvers, MA, USA) and β-actin as a loading control (Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4 °C. Specific bands were visualized using ECL Western Blotting Detection System (Amersham Biosciences, Buckinghamshire, UK). Subsequently, the intensity of the bands was quantified through densitometry analysis using Image J software (version 1.52p) by measuring the integrated optical density (IOD).
6
Sulforaphane Modulates NF-κB Activity
7
Western Blot Analysis of Oxidative Stress Markers
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Total proteins were separated by SDS-PAGE, transferred on to PVDF membranes, and incubated with the following primary antibodies: anti-iNOS (1 : 1000, ab178945, Abcam), anti-p65NF-Κb (1 : 1000, 8242S, Cell Signaling Technology), anti-IκBα (1 : 1000, 4812S, Cell Signaling Technology), anti-p-IκBα (1 : 1000, 2859S, Cell Signaling Technology), anti-Histone (1 : 1000, 4499S, Cell Signaling Technology), anti-VCAM-1(1 : 1000, ab134047, Abcam), anti-ICAM-1 (1 : 1000, ab119871, Abcam), anti-COX-2 (1 : 1000, 12282S, Cell Signaling Technology), anti-cleaved PARP (1 : 1000, 5625S, Cell Signaling Technology), anti-cleaved caspase-3 (1 : 1000, 9661S, Cell Signaling Technology), anti-Bax(1 : 1000, 2772S, Cell Signaling Technology), anti-Bcl2(1 : 1000, 3498S, Cell Signaling Technology), anti-Akt (1 : 1000, 4691S, Cell Signaling Technology), anti-phospho-Akt (p-AKT) (1 : 1000, 4060S, Cell Signaling Technology), anti-Nrf2 (1 : 1000, 12721S, Cell Signaling Technology), anti-HO-1 (1 : 1000, 70081S, Cell Signaling Technology), and anti-β-actin (1 : 5000, A1978, Sigma). After further incubation with corresponding secondary antibodies, signals were detected with a chemiluminescent reagent.
8
Lung Tissue Culture Protocol for Adenocarcinoma Research
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The study was approved by our institutional Ethics Committee (Protocol number CRC 05-088 S1R2). Human lung explants were obtained from 4 patients aged between 50-60 years undergoing surgery for lung carcinoma resection. Tissues samples were collected from fresh lobectomy and transported to the laboratory in physiological Krebs’ solution (Sosroseno and Sugiatno, 2008 (link)). Human lung adenocarcinoma cell line (A549 cells) was from Abcam (# ab7910). NCS 613 (patent FR0601958) was given by J.J. Bourguignon and C. Lugnier (Faculty of pharmacy, Strasbourg). PDE antibodies used for Western blot analysis and immuno-staining were from FabGennix Inc. Anti-phospho and total ERK1/2, anti-IκBα, anti-p65-NF-κB, Anti-phospho, and total p38-MAPK were from Cell Signaling Technology. [3H]-thymidine (250 μCi) was obtained from New England Nuclear.
9
LPS-Induced Signaling Pathway Analysis
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The cells were cultured in αMEM or rDFSC-CM with 0.5 mg/L LPS for 0, 15, 30, 60, and 120 min, and protein was then collected by lysis with phosphatase inhibitor and protease inhibitor (Thermo Fisher Scientific, USA). The protein contents were quantified using a bicinchoninic acid protein assay kit (Biocolors, China). Thirty micrograms of proteins was separated on sodium dodecyl sulfate-polyacrylamide gels and transferred to nitrocellulose membranes (Millipore, USA). The membranes were probed with the following primary antibodies overnight at 4 °C: anti-p-p38 MAPK, anti-p38 MAPK, anti-p-ERK 1/2, anti-ERK1/2, anti-p-SAPK/JNK, anti-SAPK/JNK, anti-p-p65 NF-κB, anti-p65 NF-κB (1:1000, Cell Signaling Technology, USA), and anti-vinculin (1:5000, Abcam, USA). The membranes were subsequently washed for 30 min and incubated with horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology, USA) at 1:5000 dilution. The immunoreactive proteins were visualized by enhanced chemiluminescence (ECL; Millipore, USA), and the densities of the protein bands were quantified using ImageJ version 1.50i software (Bethesda, USA).
10
Immunoblotting for Cytoplasmic Protein Analysis
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Cytoplasmic proteins were prepared from splenic tissue and immunoblot analysis was performed as described [6] . Briefly, total proteins were extracted with the use of RIPA lysis buffer (Pierce Biotechnology, Rockford, Ill., USA). Samples containing equal amounts of protein were separated by 10% SDS-PAGE and transferred onto Hybond TM ECL membranes (Amersham Pharmacia Biotech, Piscataway, N.J., USA), which were incubated overnight at 4 ° C with the appropriate primary antibodies (anti-phospho-p38 MAPK, anti-p38 MAPK, anti-phosphor-p65 NF-κB, anti-p65 NF-κB, anti-phosphor-JNK, anti-JNK (Cell Signaling Technology, Beverly, Mass., USA; 1: 1,000), and then incubated for 1 h at room temperature with peroxidase-conjugated secondary antibodies (Cell Signaling Technology; 1: 5,000). Blots were exposed to the SuperSignal West Dura Extended Duration Substrate (Pierce Biotechnology). Signals were quantified by scanning densitometry with the Bioimage analysis system (Bio-Rad).
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