Melatonin was purchased from J&K, Chemical Ltd., and epirubicin was purchased from Pfizer, pharmaceutical Ltd. Melatonin and epirubicin were dissolved in dimethyl sulphoxide (DMSO) and made into stock solution before adding into the complete culture medium. The maximum concentration of DMSO in the culture medium did not exceed 0.1%. The primary antibodies for GAPDH, LaminB1, and P-glycoprotein were purchased from Proteintech (Proteintech, Inc., USA). The antibodies for Bcl-2, cleaved-PARP, IKKβ, p-IKKα/β, NF-κB P50, and P65 were purchased from Cell Signaling Technology (Cell Signaling Technology, Inc., USA). The antibody for cytochrome c was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Nf κb p50
Manufactured by Cell Signaling Technology
Sourced in United States
NF-κB p50 is a core subunit of the NF-κB transcription factor complex. It plays a key role in regulating gene expression involved in immune and inflammatory responses.
Automatically generated - may contain errors
Lab products found in correlation
Nf κb p65, by Cell Signaling Technology (5 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Melatonin, by J&K Scientific (2 mentions)
Cleaved parp, by Cell Signaling Technology (2 mentions)
Bcl 2, by Cell Signaling Technology (2 mentions)
P ikkα β, by Cell Signaling Technology (2 mentions)
Mmp 9, by Santa Cruz Biotechnology (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Lamin b1, by Proteintech (1 mentions)
P glycoprotein, by Proteintech (1 mentions)
Epirubicin, by Pfizer (1 mentions)
Anti mmp2, by Cell Signaling Technology (1 mentions)
Anti p p38, by Cell Signaling Technology (1 mentions)
Nf κb p52, by Cell Signaling Technology (1 mentions)
Mmp 9, by Cell Signaling Technology (1 mentions)
Anti e cadherin, by Abcam (1 mentions)
Anti vimentin, by Abcam (1 mentions)
Rabbit secondary antibody, by Cell Signaling Technology (1 mentions)
Analytical grade organic solvents, by Merck Group (1 mentions)
Nucleolin, by Cell Signaling Technology (1 mentions)
11 protocols using nf κb p50
1
Melatonin and Epirubicin Anticancer Effects
2
Evaluation of EMT Markers and NF-κB Signaling
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Anti E-cadherin and Anti Vimentin were purchased from Abcam. Fetal bovine serum (FBS), penicillin, streptomycin and Dulbecco's modified Eagle's medium (DMEM) were ordered from Hyclone. Anti-MMP-2, MMP-9, NF-κB p50, NF-κB p65, NF-κB p52, NF-κB RelB, NF-κB c-Rel anti-p38 and anti-p-p38 antibodies were purchased from Cell Signaling. Anti-Histone H1antibody was purchased from Santa Crus.
3
Standardized Western Blot Protocol
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Fetal bovine serum (FBS) was purchased from Gibco/BRL (Burlington, ON, Canada). Pen Strep was purchased from life technologies corporation, Grand Island, NY, USA. Enhanced chemiluminescence (ECL) regent was purchased from Amersham, Arlington Heights, IL, USA. The enzyme linked immunosorbent assay kit (ELISA) for prostaglandin E2 (PGE2) (cat# KGE004B) and IL-1β (cat# MLB00C) were purchased from R&D System Inc. (Minneapolis, MN, USA). ELISA kits for mouse TNF-α (cat# 560478), and IL-6 (cat# 550950) were purchased from Becton, Dickinson and Company (BD Biosciences; San Jose, CA, USA). Cytosolic and nucleus proteins extracted from commercial protein extraction kit (#78833) were purchased from Thermo Scientific; Rockford, USA. The following primary antibodies for Western blots: iNOS (#13120), COX2 (#12282), phospo NF-κB-P50 (#4806), NF-κB-P50 (#3035), phospo NF-κB-P65 (#3033), NF-κB-P65 (#8242), nucleolin (#14574), β-actin (#4970), and rabbit secondary antibody (#7074) were purchased from Cell Signaling Technology MA, USA). All analytical grade organic solvents were purchased from Sigma-Aldrich or otherwise mentioned in the text.
4
Protein Analysis of Tumor Cell Signaling
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Proteins from tumor tissue or cultured cells were extracted with ice-cold radioimmunoprecipitation assay (RIPA) buffer and subjected to SDS-PAGE and then electro-transfer to nitrocellulose membranes. After blocking with 5% BSA/Tris-buffered saline (TBS) buffer, membranes were incubated with primary antibodies against mouse p-Smad3, Smad7, p-ALK5 (Abcam, MA, USA), MMP2, MT1-MMP, TIMP2 (Merck Millipore, MA, USA), MMP9, MMP13, β-actin (Santa Cruz Biotechnology, CA, USA), and NF-κB p50 (Cell Signaling, MA, USA) dissolved in 1% BSA overnight at 4°C, followed by incubating with IRDye 800-conjugated secondary antibody (Rockland Immunochemicals, PA, USA). Signals of target proteins were detected by Li-Cor/Odyssey infrared image system (LI-COR Biosciences, NE, USA) and then analyzed with ImageJ software (NIH, Bethesda, MD, USA).
5
LPS-Induced Inflammation Modulation
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Mangiferin (MF), purity>99.84%, was purchased from the Shanghai Winherb Medical Co. (Shanghai, China). Lipopolysaccharide (LPS) (Escherichia coli 055:B5) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Anti-β-actin (ab6276), anti-NLRP3 (ab263899), anti-ASC (ab151700) and anti-GSDMD (ab219800) were obtained from Abcam (Cambridge, MA, USA). Anti-NF-κB p65 (#8242) and NF-κB p50 (#3035) were provided by Cell Signalling Technology (Danvers, MA, USA). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were obtained from Invitrogen-Gibco (Grand Island, NY). The secondary antibody was provided by LI-COR Biosciences (Lincoln, United States). All chemicals used in this study were of analytical grade.
6
Antibody Panel for Cell Signaling Pathway Analysis
7
Cell Signaling Pathway Analysis
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Dimethyl sulfoxide (DMSO), 3-(4-5-dimethyl-2yl)-2-5-diphynyltetrasolium bromide (MTT), collagenase, azo dye-impregnated collagen, and 2′, 7- dichlorodihydrofluorescein diacetate (DCFH2-DA) were purchased from Sigma Co. (St. Louis, MO, USA). Phosphate-buffered saline (PBS), penicillin/streptomycin (P/S), Dulbecco’s modified Eagle’s medium (DMEM), F-12 medium, and fetal bovine serum (FBS) were purchased from Gibco BRL (Life Technologies, Burlington, ON, Canada). Antibodies against p-p38, p-JNK, p-ERK, GAPDH, p-c-Jun, nucleolin, NF-κB p65, and NF-κB p50 were purchased from Cell Signaling Technology (Beverly, MA, USA). The ELISA kits used for analysis of human MMPs, interleukin-6 (IL-6), interleukin-1 beta (IL-1β), and tumor necrosis factor-alpha (TNF-α) were purchased from GE Healthcare Life Sciences (Exeter, Devon, UK). The PIP EIA kit was purchased from TaKaRa Bio Inc. (Kusatsu, Japan). All other chemicals used in this study were of analytical grade.
8
Probing the mTOR Signaling Pathway
9
Combination of Melatonin and Vemurafenib
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Melatonin was purchased from J&K, Chemical Ltd. Vemurafenib was obtained from Selleck (PLX4032) and dissolved in dimethyl sulphoxide (DMSO) before addition to the complete cell culture medium. For the experiment, the solution of Melatonin (1 M) and Vemurafenib (VE) (10 mM) in DMSO was prepared and kept at 4 °C for further dilution in culture medium to maintain stability of used drugs. The InSolution™ NF-κB Activation Inhibitor controlling the biological activity of NF-κB (481407) was bought from Merck Millipore. Antibodies against β-catenin, MMP-1, MMP-9, Apaf-1 were purchased from Santa Cruz (USA). The antibodies against β-actin, IKKα, IKKβ, p-IKKα/β, IκB-α, p-IκB-α, cleaved caspase-3, cleaved caspase-9, cleaved PARP, Bcl-2, NF-κB p50, p65, p-PDK1, p-PTEN, p-AKT and AKT were purchased from Cell Signaling Technology (USA). The anti-TFIIB, E-cadherin antibodies were purchased from Proteintech group (USA), and anti-iNOS antibody was purchased from Wanlei Biotechnology (China). The anti-BRAF V600E antibody was purchased from Omnimabs (USA).
10
Isolation and Analysis of Nuclear Proteins
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T cells were collected and lysed with nuclear isolation buffer (10 mM Hepes-KOH, pH 8.0, 10 mM KCl, 1.5 mM MgCl2, 0.34 M sucrose, 10% glycerol, 1 mM dithiothreitol, and 1× protease inhibitors) for 10 min with shaking every 3 min. The nuclear pellet was collected by centrifugation and was suspended and washed with nuclear isolation buffer twice. The nuclear pellet was directly lysed in SDS sample buffer with 8 M urea. SDS-PAGE was performed in the Bolt Bis-Tris system (Thermo Fisher Scientific). Transfer was performed with 120 V for 90 min to a polyvinylidene difluoride membrane. Blocking of the membrane was performed with Odyssey Blocking Buffer (#927-50000; LI-COR) for 60 min at RT with shaking. The antibody incubation was performed at 4°C overnight with an orbital shaker. Antibodies to cFOS (#2250), JUNB (#3753), NFAT1 (#5861), NFAT2 (#8032), NF-κB p50 (#12540), and PARP1 (#9532) from Cell Signaling; NF-κB p65 (c15310256) from Diagenode; and GAPDH (NB600-502) from Novus were used as primary antibodies. Membranes were washed with Tris-buffered saline, pH 7.5, 0.1% Tween for 5 min three times and were incubated with IRDye secondary antibodies in 1/15,000 dilution in blocking buffer for 60 min at RT with shaking. Membranes were washed with TBS-Tween for 5 min three times again and with TBS once. Images were taken with an Odyssey Fluorescent Imaging system (LI-COR).
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