In these experimental studies, human A549, HEK293T and Kyse150 cell lines (Chinese Academy of science, shanghai, China) were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco) supplemented with 10% fetal bovine serum, 1% glutamine, and 1% penicillin–streptomycin. All cells were maintained in a humidified atmosphere of 5% CO2 at 37°C. The antibodies of TXNIP, TRAF6, Caspase‐1/3, Bax, GAPDH, Tubulin, and beta‐Actin were purchased from Abcam Trading (Shanghai) Company (China). Anti‐Flag, anti‐HA antibodies, MG132 were obtained from Sigma. TRIzol and cDNA synthesis kit were from Invitrogen. The second antibodies were obtained from Biorad. Other antibodies were purchased from Cell Signaling Technology. Dual luciferase reporter assay kit was obtained from Promega. Lipofectamine® 3000 was purchased from Thermo Fisher Scientific. All other reagents were obtained from Shanghai Shenggong, China or Sigma. For reagent treatment, cells were loaded onto a culture dish 1 day before treatment with designated time points and concentrations.
Traf6
Manufactured by Abcam
Sourced in United States, United Kingdom
TRAF6 is a protein that plays a key role in the activation of nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways. It functions as an E3 ubiquitin ligase, catalyzing the formation of Lys63-linked polyubiquitin chains, which are important for signal transduction.
Automatically generated - may contain errors
Lab products found in correlation
Myd88, by Abcam (10 mentions)
β actin, by Abcam (8 mentions)
Pvdf membrane, by Merck Group (6 mentions)
β actin, by Santa Cruz Biotechnology (5 mentions)
Tris glycine gel, by Thermo Fisher Scientific (4 mentions)
Tnf α, by Abcam (4 mentions)
Bcl2 associated x (bax), by Abcam (3 mentions)
Irak1, by Abcam (3 mentions)
Il 1β, by Abcam (3 mentions)
Epac1, by Abcam (3 mentions)
Bca protein assay kit, by Beyotime (3 mentions)
P nf κb p65, by Abcam (3 mentions)
Nf κb p65, by Abcam (3 mentions)
Nf κb p65, by Cell Signaling Technology (3 mentions)
Gapdh, by Abcam (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Chemilluminescence reagent kit, by Thermo Fisher Scientific (2 mentions)
Azure c500 machine, by Azure Biosystems (2 mentions)
P akt, by Cell Signaling Technology (2 mentions)
Nfatc1, by Santa Cruz Biotechnology (2 mentions)
40 protocols using traf6
1
Cell Culture Protocols for A549, HEK293T, and Kyse150
2
Protein Expression Analysis of NP Cells and MSCs-EVs
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The lysates of NP cells and MSCs-EVs made by radio-immunoprecipitation assay buffer (Beyotime) were detected for determining protein concentration with a bicinchoninic acid protein analysis kit II (BIO-RAD, Hercules, CA, USA). Protein samples were separated by 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis and electroblotted onto nitrocellulose membranes (Invitrogen). Reacted with the CD9 (1:1000, Santa Cruz Biotechnology), CD81 (1:1000, Santa Cruz Biotechnology), Caspase-3 (1:1000, Cell Signaling Technology), Cleaved Caspase-3 (1:1000, Cell Signaling Technology), TRAF6 (1:1000, Abcam) and GAPDH (1:1000, Cell Signaling Technology) overnight, the protein samples were probed with horseradish peroxidase-conjugated secondary antibodies. The signals on the membrane were observed with enhanced chemiluminescence reagent (Millipore) and detected by Amersham™ Imager 680 (GE Healthcare Life Sciences) [22 (link)].
3
Protein Expression Analysis by Western Blotting
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Protein extraction and western blot analysis was performed as described previously [47 (link)]. Briefly, proteins were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. After non-specific sites were blocked with 5% non-fat milk, the membranes were incubated with antibodies against Ki67(Abcam, Cambridge, UK), PCNA (Cell Signaling Technology, Danvers, MA, USA), Bcl-2 (Santa Cruz Biotech, Santa Cruz, CA, USA), Bax (Santa Cruz Biotech), phospho-caspase-3 (Santa Cruz Biotech), TLR4 (Sigma-Aldrich), MyD88 (Abcam), TRAF6 (Abcam), ERK1 (Santa Cruz Biotech), phospho-ERK (Thr202/Tyr204; Santa Cruz Biotech), JNK (Santa Cruz Biotech), phospho-JNK (Thr183/Tyr185; Santa Cruz Biotech), p38 (Santa Cruz Biotech), and phospho-p38 (Thr180/Tyr182; Santa Cruz Biotech) and β-actin (Sigma-Aldrich) at 4°C overnight. The membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature, and blots were visualized using the Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA). β-actin was used as an internal control.
4
Extracellular Vesicle Protein Analysis
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Protein extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (0.22 μm, Millipore, Massachusetts, USA). The membranes were then blocked with 5% skimmed milk for 1.5 hours. Secondary antibodies were then added for cultivation. An electrochemiluminescence system was subsequently used for the detection of the immunoreactive bands.
The antibodies included were anti-CD63 (Abcam, Cambridge, UK), anti-Alix (Abcam, USA), anti-CD 81 antibody (Abcam, UK), microtubule associated protein 1 light chain 3 beta (anti-LC3B) (Abcam, UK), anti-p62 (Abcam, UK), anti-Caspase-3 (Abcam, UK), anti-Bcl-2 (Abcam, UK), tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) (Abcam, UK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Abcam, UK) horseradish peroxidase-conjugated goat anti-rabbit IgG (Proteintech, Wuhan, China), and horseradish peroxidase-conjugated goat anti-mouse immunoglobin (IgG) (Proteintech, Wuhan, China).
The antibodies included were anti-CD63 (Abcam, Cambridge, UK), anti-Alix (Abcam, USA), anti-CD 81 antibody (Abcam, UK), microtubule associated protein 1 light chain 3 beta (anti-LC3B) (Abcam, UK), anti-p62 (Abcam, UK), anti-Caspase-3 (Abcam, UK), anti-Bcl-2 (Abcam, UK), tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) (Abcam, UK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Abcam, UK) horseradish peroxidase-conjugated goat anti-rabbit IgG (Proteintech, Wuhan, China), and horseradish peroxidase-conjugated goat anti-mouse immunoglobin (IgG) (Proteintech, Wuhan, China).
5
Analyzing Gadus morhua Egg Signaling
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The Gadus morhua eggs were purchased from Guangzhou Meiyu Food Co., Ltd., (Guangzhou, China). TRAF6, OPG, RANKL, and β-actin antibodies were from Abcam (Cambridgeshire, United Kingdom). The primers of TRAF6, RANKL, OPG, and GADPH were synthesized by ShengGong Ltd., Co. (Shanghai, China). All chemicals and reagents used were of analytical grade.
6
Western Blot Analysis of TLR4 Signaling
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Retinal lysates from untreated TLR4 floxed and TLR4 Cre-Lox mice were rinsed with cold PBS, collected in lysis buffer containing protease and phosphatase inhibitors, and scraped into tubes. Equal amounts of protein were separated on precast tris-glycine gels (Invitrogen, Carlsbad, CA), and then blotted onto a nitrocellulose membrane. After blocking in TBST (10mM Tris-HCl buffer, pH 8.0, 150 mM NaCl, 0.1% Tween 20) and 5% (w/v) BSA, the membrane was treated with appropriate primary antibodies followed by incubation with secondary antibodies labeled with horseradish peroxidase. Antigen-antibody complexes were detected by chemilluminescence reagent kit (Thermo Scientific, Pittsburgh, PA). Primary antibodies used were TLR4, MyD88, TRIF, TRAF6, IRAK1, and IRF3 (Abcam, Cambridge, MA) and beta actin (Santa Cruz, Santa Cruz, CA).
7
Western Blot Analysis of Retinal Signaling
8
Chondrocyte Differentiation Assay
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The cell-culture reagents were provided by Gibco BRL. Recombinant murine sRANKL (#315–11) and Recombinant murine HGF (#315–23) were purchased from PeproTech. Recombinant mouse MCSF protein (#ab129146), and the following antibodies: p65 (#ab16502), TRAF6 (#ab33915), JNK (#ab124956), HGF (#ab83760), were obtained from Abcam. The following antibodies were purchased from Cell Signaling Technology: p-p65 (#3033), p-JNK (#9255), Met (#8198), p-Met (#3077), Akt (#4685), p-Akt (#4060), GSK-3β (#9315S), p-GSK-3β (#9323). NFATc1 (#sc-17834) and GAPDH (#sc-32233) antibodies were provided by Santa Cruz Biotechnology. β-actin (#60008-1-Ig) and Histone-3 (#17168-1-AP) antibodies were purchased from Proteintech Group. SU11274 was obtained from Selleck. Immunization Grade Chick type II collagen (#20012), Complete Freund’s Adjuvant (#7001), and InComplete Freund’s Adjuvant (#7002) were purchased from Chondrex.
9
Apoptosis and DNA Damage Assessment
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The FITC-labelled Annexin V Apoptosis Detection Kit was obtained from BD Biosciences (San Jose, CA, USA). DNA Damage Quantification Colorimetric Kit was purchased from Biovision (Milpitas, CA, USA) and EpiQuik In Situ DNA Damage Assay kit was obtained from Epigentek (Farmingdale, NY, USA). JC-1, cisplatin, and DCFH-DA were purchased from Sigma-Aldrich (St. Louis, MO, USA). The culture medium was obtained from Invitrogen Technologies (Carlsbad, CA, USA). The antibodies were obtained from the following sources: TRAF6 and MVP from Abcam (Cambridge, UK); α-tubulin from Sigma-Aldrich; p62, NFκB, cleaved-PARP, cleaved-caspase 3, cleaved-caspase 7, cleaved-caspase 9, IKKβ, IKKα, phosphor-IKKβ, horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody, HRP-conjugated goat anti-mouse antibody, and Alexa Fluor 488-conjugated goat anti-mouse antibody from Cell Signalling Technology (Danvers, MA, USA).
10
Pig Model of Respiratory Infection
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HD-13 was provided by China Agricultural University (Beijing, China), which was provided ready to use. The HD-13 (104 CFU/mL) strain was infected by nasal drip and respiratory tract infection, and the bacterial solution was dropped into the nasal cavity of pigs. Reagents, including PD98059, which served as the p-ERK/ERK signaling pathway inhibitor, SB203580 which served as the p-p38/p38 signaling pathway inhibitor, SP600125 which served as the p-JNK/JNK signaling pathway inhibitor, and BAY11-7082 which served as the NF-κB signaling pathway inhibitor, were purchased from Solarbio (Beijing, China). Antibodies, including TLR9, MyD88, TRIF, TRAF6, p-ERK, ERK, p-p38, p38, p-JNK, JNK, p-p65, p65, p-IKKα, IKKα, and β-actin, were provided by Abcam (Cambridge, UK, 1 : 1000, monoclonal, species of mice).
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