FBS, Horse Serum, antibiotic/antimycotic, EGF, insulin, Cholera Toxin, Hydrocortizone, L-Glutamine, HEPES, Sodium Pyruvate and media used in cell culture were purchased from Gibco, Invitrogen (Carlsbad, California). Etoposide and Doxorubicin were purchased from Tocris (Bristol, UK). The pan caspase inhibitor z.vad.fmk was purchased from Sigma (St. Louis, Missouri). TNFα, and PS-341 (Bortezomib) were purchased from Merck Millipore (Darmstadt, Germany). Protease inhibitor cocktail was obtained from Roche (Indianapolis, IN). Caspase −3, −7, −8, −9, PARP1, RIP1, Bid, TNFR1, TNFR2, AIF, FADD, p-IκBα, p-TRAF2, p65/NF-κB and α-Tubulin antibodies were purchased from Cell Signaling Technology (Danvers, Massachusetts). Total TRAF2, total IκBα, Histone H3, EGFR, and GAPDH antibodies were obtained from Santa Cruz Biotechnology (Heidelberg, Germany). All other reagents were purchased from Sigma (St. Louis, Missouri).
Tnfr2
Manufactured by Cell Signaling Technology
Sourced in United States
TNFR2 is a cell surface receptor that binds to the cytokine tumor necrosis factor-alpha (TNF-alpha). It mediates cellular responses to TNF-alpha signaling.
Automatically generated - may contain errors
Lab products found in correlation
Tnfr1, by Cell Signaling Technology (4 mentions)
Caspase 3, by Cell Signaling Technology (4 mentions)
Caspase 9, by Cell Signaling Technology (3 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (3 mentions)
Bcl 2, by Cell Signaling Technology (3 mentions)
Caspase 8, by Cell Signaling Technology (3 mentions)
α tubulin, by Cell Signaling Technology (2 mentions)
Gapdh, by Santa Cruz Biotechnology (2 mentions)
Tnf α, by Santa Cruz Biotechnology (2 mentions)
P p38 mapk, by Cell Signaling Technology (2 mentions)
Sirt3, by Cell Signaling Technology (2 mentions)
P jnk, by Cell Signaling Technology (2 mentions)
P erk, by Cell Signaling Technology (2 mentions)
β actin, by Merck Group (2 mentions)
Traf2, by Cell Signaling Technology (2 mentions)
Ecl kit, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Etoposide, by Bio-Techne (1 mentions)
Doxorubicin, by Bio-Techne (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
9 protocols using tnfr2
1
Cell Signaling Pathway Analysis
2
Evaluating Apoptosis-Inducing Pathways
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Without specific indication, the reagents purchased from Sigma‐Aldrich Inc. were used in the present study, and cell culture supplements were from GIBCO/Life Technologies Inc. Vincristine (VCR), SZLP1‐41 (Skp2 inhibitor), E64D (lysosomal protease inhibitor) and epoxomicin (proteasome inhibitor) were the products of Apexbio Technology LLC. GKT137831 and GLX351322 were purchased from MedChem Express; MitoSOX, annexin V‐FITC/propidium iodide (PI) apoptosis detection kit, tetramethylrhodamine (TMRM) and H2DCFDA were from Molecular Probes (Eugene, OR). Antibodies against caspase‐3 and caspase‐8, okadaic acid, Z‐IETD‐FMK and Z‐DEVD‐FMK were purchased from Calbiochem. Antibodies separately against PP2Ac, Mcl‐1, tristetraprolin (TTP), Fas, FasL, TNF‐α and FADD were purchased from Santa Cruz Biotechnology Inc.; and antibody against TNFR1 was from R&D Systems. Antibodies separately against ERK, p‐ERK, p38 MAPK, p‐p38 MAPK, JNK, p‐JNK, SIRT3, TNFR2, α‐tubulin, MID1, α4, caspase‐9, Bax, Bak and Bcl‐2 were obtained from Cell Signaling Technology. Antibodies separately against cytochrome c, Bcl‐xL and Bid were the products of BD Pharmingen Technical (San Jose, CA), and anti‐NOX4 antibody was from Novus Biologicals. Secondary antibodies conjugated with horseradish peroxidase (HRP) were the products of Pierce (Rockford, IL).
3
Comprehensive Immunoblotting Analysis Protocol
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Immunoblotting was performed essentially as previously described [68 (link)]. The primary antibodies used for immunoblotting were shown as following: β-actin (Sigma-Aldrich Inc., St. Louis, MO, USA), BCL2L1, BID (BD Pharmingen Technical, San Jose, CA, USA), BAX, BAK, BCL2, SIRT3, TNFR2, DR4, DR5, JNK, p38 MAPK, ERK, p-JNK, p-p38 MAPK, p-ERK, caspase-3, caspase-8 (Cell Signaling Technology, Beverly, MA, USA), NOX4 (Novus Biologicals, Centennial, CO, USA), FADD, TNF-α, FasL, Fas, PP2Acα, MCL1, tristetraprolin (TTP), PARP, TRAIL (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and TNFR1 (R&D Systems, Minneapolis, MI, USA). Protein bands visualization was done with enhanced chemiluminescence substrate (Perkin Elmer. Waltham, MA, USA). Each immunoblot was performed no less than three times.
4
Inflammatory Signaling Pathway Modulation
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Diclofenac and ibuprofen were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and dissolved in dimethyl sulfoxide as 10 mM stock solution, stored at −20 °C. Propidium iodide was purchased from AppliChem (Darmstadt, Germany) and 3,3′-dihexyloxacarbocyanine iodide (DiOC6) from Calbiochem (La Jolla, CA, USA). 4′,6-Diamidino-2-fenilindol (DAPI) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti–rabbit antibodies against p53, Mouse double minute 2 (Mdm-2), AKT, FoxO1, FoxO3a, Fas, caspase-8, caspase-7, caspase-2, RIP, TNF-R2, procaspase-9, procaspase-3, Bcl-2, Mcl-1, Bcl-x, Bax, BimEL, Puma, Bak, Bid, p38, SAPK/JNK, p-SAPK/JNK, Axin, Wnt5a/b, Dvl3, Dvl2, β-catenin, Wnt3a, LRP6, E-cadherin, N-cadherin, vimentin, Snail, Lamin A/C, GAPDH, and β-actin (each 1:500-1:1000) were bought from Cell Signalling Technology (CST, Danvers, MA, USA). Horseradish peroxidase–conjugated secondary anti–rabbit antibodies or anti–mouse antibodies (1:5000) were obtained from CST.
5
HL-60 Cells Cytotoxicity Assay
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Methylthiazolyldiphenyl-tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), and propidium iodine (PI) were purchased from Sigma-Aldrich (St. Louis, MO, USA). JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′ Tetra- ethylbenzimidazolylcarbocyanine iodide) was obtained from Molecular Probes (Invitrogen, Karlsruhe, Germany). Q-VD-OPh was purchased from R&D (Minneapolis, MN, USA). The antibodies of GAPDH, actin, PARP-1, and Rad51were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Antibodies of DFF45/DFF35, TNFR1, TNFR2, Fas, DR5, Bid, cleaved caspase-1, caspase-3, caspase-5, caspase-8, gasdermin D, cIAP1, cIAP2, survivin, HMGB1, and γH2A.XSer139 were obtained from Cell Signaling Technologies (Boston, MA, USA). Antimouse and antirabbit IgGs were obtained from Jackson Immuno-Research Laboratories, Inc. (West Grove, PA, USA). HL-60 (promyelocytic leukemia) was obtained from Bioresource Collection and Research Center (BCRC, Hsinchu, Taiwan). HL-60 cells were cultured in Iscove’s Modified Dulbecco’s Medium (IMDM) (Gibco, Grand Island, NY, USA) with 10% FBS (v/v) and penicillin (100 U/mL)/streptomycin (100 mg/mL). The cells were grown in a water-saturated atmosphere at 5% CO2 and at 37 °C.
6
Investigating Cell Signaling Pathways in Apoptosis
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Dimethylsulfoxide (DMSO), 3-(4,5-dimethylthylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), and all other chemicals were purchased form Sigma-Aldrich (St. Louis, MO, USA). Antibodies against PARP, caspase-3, caspase-8, caspase-9, Bak, IRE1α, PERK, TNFR2, CDK6, p-cdc2, p-Rb (ser795), p-ATM, p-ATR, p-Chk1, and p-Chk2 were purchased from Cell Signaling Technologies (Beverly, MA, USA). Antibodies of Bcl-2, LC3, NF-κB, p-H2AX, ATF6, and CDK4 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies of TNFR1, RIPK1, RIPK3, and MLKL were purchased from ABclonal (ABclonal, Wuhan, China). Antibodies of Actin and p62/SQSTM1 were purchased from Sigma (Sigma, CA, USA). Anti-mouse and rabbit IgG peroxidase-conjugated secondary antibody were purchased from Pierce (Rockford, IL, USA). Hybond ECL transfer membrane and ECL Western blotting detection kits were purchased from Ameisham Life Sciences (Amersham, Buckinghamshire, UK). Rhodamine 123 and the carboxy derivative of fluorescein (carboxy-H2DCFDA) were purchased from Molecular Probes and Invitrogen detection technologies (Carlsbad, CA, USA).
7
Analyzing Apoptosis Signaling Pathways
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Primary antibodies for poly(ADP-ribose) polymerase (PARP), caspase-3, caspase-7, cleaved caspase-8, caspase-9, phospho-Bad (p-Bad) (Ser112), Bid, Bax, Fas, TNF-R1, TNF-R2, DR4, and anti-mouse or anti-rabbit secondary antibody horse radish peroxidase (HRP) conjugates were purchased from Cell Signaling Technology (Beverly, MA). TRAILsiRNA and β-actin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Amaxa Cell Line Nucleofector kit was purchased from Lonza (Lonza, Basel, Switzerland). The BCA protein assay kit and SuperSignal West Pico chemiluminescent substrate kit were purchased from Pierce (Pierce, Thermo Scientific, Rockford, IL). The mini-protean precast Tris-Glycine gels were purchased from BioRad (Hercules, CA). Anti-mouse or anti-rabbit secondary antibody biotinylated conjugates were obtained from Vector Laboratories (Burlingame, CA). Annexin-V-FLUOS staining kit was purchased from Roche Diagnostic Corporation (Indianapolis, IN). NF-κB Activation Inhibitor IV was purchased from Calbiochem (EMD Millipore Corporation, Billerica, MA).
8
Comprehensive Immunoblotting Protocol
9
Western Blot Analysis of TNFR2 Signaling
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Treated NCI-N87 cells (5 × 10 5 ) were harvested and lysed using RIPA buffer (Thermo Scientific, USA) for 10 min at 4 °C. BSA Standard Solution (2 mg/ml) from BCA Protein Assay Kit (Tiangen, China) was diluted at a gradient concentration for standard curve construction according to the instructions. The blotting process was performed at eZwest Lite Auto Western Blotting System (Nanjing, China). Equivalent amount of protein (20 μg) was separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis, and then transferred to polyvinylidene difluoride membranes (Millipore, USA). The membranes were blocked with skimmed milk (5%) for 1 h. Then, the membranes were incubated with the primary antibodies against TNFR2 (catalog #3727, Cell signaling, USA), TRAF2 (#4712, Cell signaling), PI3K (#4255, Cell signaling), p-PI3K (#4228S, Cell signaling), AKT (#4060, Cell signaling), p-AKT (#4060, Cell signaling) , IκBα, and p-IκBα (all 2 μg/ml), overnight at 4 °C, with β-actin (clone #SP124, Abcam, USA) as control. Then, biotinylated goat anti-rabbit IgG (#A0277; 2 μg/ml; Beyotime Biotech) was added. Horseradish peroxidase-streptavidin and an ECL kit (Thermo Scientific, Waltham, MA, USA) were used for color rendering. The band intensities were quantified using Image J software. The quantification of each immunoblot was performed on three biological repeats.
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