The normal human bronchial epithelioid cell (HBE) and the non-small-cell lung cancer cell lines including NCI-H1650, NCI-H1299, A549, NCI-H460, HCC4006, NCI-H1975, and NCI-H358 were obtained from ATCC and cultured with recommended culture medium. The primary antibodies for western blotting including anti-TRAF6 (#8028), hexokinase-1 (#2024), hexokinase-2 (#2867), GLUT1 (#12939), PKM2 (#4053), LDHA (#35 82), VDAC-1 (#4661), phosphor-Akt (#4060), Akt (#8596), phosphor-S6 (#4858), and ubiquitin (#58395) as well as the secondary anti-rabbit IgG HRP (#7074) were products of Cell Signaling Technology Inc. (Danvers, MA). β-Actin (A5316) was obtained from Sigma-Aldrich. In immunohistochemistry staining, the primary antibodies against hexokinase-2 (ab227198) and Ki67 (ab15580) were products of Abcam. TRAF6 shRNA#1 (TRCN0000007350) and shRNA#2 (TRCN0000007351) were purchased from the Sigma Mission shRNA library. The constitutively active Akt (CA-Akt) plasmid (Cat. #10841) and pLKO.1 GFP shRNA (Cat. #30323) were purchased from Addgene (Cambridge, MA, USA). Recombinant human insulin-like growth factor 1 (IGF-1) was a product of R&D (Cat. 291-G1-200). Lipofectamine 2000 was obtained from Invitrogen (Carlsbad, CA).
Anti traf6
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-TRAF6 is a laboratory reagent that specifically binds to and detects the TRAF6 protein. TRAF6 is an important signaling adaptor that mediates various cellular pathways. This product can be used for the detection and analysis of TRAF6 in experimental settings.
Automatically generated - may contain errors
Lab products found in correlation
Anti p65, by Cell Signaling Technology (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Anti irak1, by Cell Signaling Technology (3 mentions)
Anti irak2, by Cell Signaling Technology (3 mentions)
Anti rabbit igg hrp, by Cell Signaling Technology (2 mentions)
Anti iκb, by Cell Signaling Technology (2 mentions)
Polyvinyl difluoride membrane, by Merck Group (2 mentions)
Anti histone h3, by Cell Signaling Technology (2 mentions)
Anti phospho iκb, by Cell Signaling Technology (2 mentions)
Anti flag m2, by Merck Group (2 mentions)
Anti k63 linkage specific polyubiquitin, by Cell Signaling Technology (2 mentions)
Anti akt, by Cell Signaling Technology (2 mentions)
Anti p38, by Cell Signaling Technology (2 mentions)
Anti jnk, by Cell Signaling Technology (2 mentions)
Anti ikkα β, by Cell Signaling Technology (2 mentions)
Anti gapdh, by Cell Signaling Technology (2 mentions)
Anti myd88, by Cell Signaling Technology (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions)
Ubiquitin, by Cell Signaling Technology (1 mentions)
15 protocols using anti traf6
1
Metabolic Reprogramming in Lung Cancer
2
Western Blot Analysis of Cell Signaling
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Cells were lysed in RIPA plus protease/phosphorylase inhibitors (Sigma) on ice. The cytosol and nuclear fraction were isolated using NE-PER Nuclear and Cytoplasmic Extraction Reagents according to the manufactory’s instruction (Thermo). Protein from each extract were separated on 4–12% SDS-PAGE and transferred to polyvinyl difluoride membranes (Millipore). After blocking with TBS plus 0.05% Tween 20 containing 5% non-fat milk for 1 h, the membranes were incubated with specific primary antibodies at 4°C overnight. Primary antibodies include anti-IL-12 p40/p35 (Santa Cruz), anti-TAB2, anti-phospho-IκB, anti-IκB, anti-p65, anti-TRAF6, anti-IRAK1, anti-IRAK2, anti-Histone H3 (Cell Signaling Technology), anti-FLAG M2 (Sigma), anti-Omp25, and anti-β-actin (Tianjin Sungene Biotech) antibodies. HRP-conjugated anti-mouse IgG or anti-rabbit IgG (Santa Cruz) were used as secondary antibodies. Pierce® Fast Western Blot Kit, ECL Substrate (Thermo) was used for chemiluminescent detection.
3
Phosphorylation-specific Beclin 1 Antibody Development
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Antibodies specific for Beclin 1 phosphorylated on Ser90 were generated by Abgent (San Diego, CA, USA, 1:500). Other primary antibodies used for western blotting were anti-Beclin 1 (Cell Signaling Technology, #3738, 1:1000), anti-GAPDH (KangChen, Shanghai, China, 1:10,000), anti-p-CaMKII (Cell Signaling Technology, #3361, 1:1000), anti-CaMKII (Santa Cruz, sc-1541, 1:500), anti-Bcl-2 (Santa Cruz, SC-7382, 1:500), anti-LC3 (Novus, Littleton, CO, USA, NB100-2220, 1:3000), anti-Id-1 (Santa Cruz, sc-488, 1:1000), anti-Id-2 (Cell Signaling Technology, #3431, 1:500), anti-SQSTM1 (Santa Cruz, sc-28359, 1:1000), anti-His-Tag (Cell Signaling Technology, #2366, 1:1000), anti-Myc-Tag (Cell Signaling Technology, #2276, 1:1000), anti-Flag (Sigma-Aldrich, F1804, 1:2000), anti-ubiquitin (Santa Cruz, sc-58449, 1:1000), anti-K63-linkage-specific polyubiquitin (Cell Signaling Technology, #5621, 1:1000), anti-TRAF-6 (Cell Signaling Technology, #8028, 1:1000), anti-GAP43 (Cell Signaling Technology, #8945 s, 1:1000), anti-NF68 (Cell Signaling Technology, #2837 s, 1:1000), anti-nestin (Santa Cruz, SC-23927, 1:1000), anti-vimentin (BD, 550513, 1:1000), and anti-E-cadherin (BD, 51-9001922, 1:1000). KN-93, MG132, and bafilomycin A1 were purchased from Sigma-Aldrich. Ionomycin was purchased from Cell Signaling Technology. EB1089 was purchased from Santa Cruz.
4
Osteoclast Differentiation Assay Protocol
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PPOA-N-Ac-2-Cl, purchased from ChemBridge (San Diego, CA, USA), was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA) to obtain a 50-mM solution. Aliquots of the solution were stored at −20 °C and diluted to the appropriate concentrations in cell culture medium immediately before use. DMSO was used as the vehicle control. Alpha-modified minimal essential medium (α-MEM) and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Bone resorption assay kit was procured from Cosmo Bio Co., Ltd., Tokyo, Japan. BCA protein assay kit was purchased from Pierce Biotechnology, (Rockford, IL, USA). Primary antibodies including anti-β-actin (Sigma-Aldrich, St Louis, MO, USA), anti-c-Src (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-cathepsin K (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-ERK1/2, anti-phospho-ERK1/2, anti-AKT, anti-phospho-AKT, anti-IκBa, anti-phospho-IκBa, anti-p38, anti-phospho-p38, anti-JNK, anti-phospho-JNK, anti-c-fos, anti-NFATc1, anti-p65, and anti-phospho-p65, anti-TRAF6, anti-calcineurinA, anti-calmodulin were purchased from Cell Signaling Technology (Boston, MA, USA). Horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from Cell Signaling Technology, and chemiluminescence signals were detected using an ECL system (iNtRON, Seoul, Korea).
5
Western Blot Analysis of Signaling Proteins
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Whole-cell protein extracts were resolved by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF; Millipore, Billerica, MA). Blots were probed with anti-NFX1-123 (1:1000), anti-p53 (1:1000, Calbiochem, San Diego, CA), (anti-TRAF6 (1:500; Cell Signaling, Danvers, MA), anti-TRIF (1:1000, Abcam, Cambridge, MA), anti-TAK1 (1:500, Abcam, Cambridge, MA), anti-TAB1 (1:500, Abcam, Cambridge, MA), anti-TAB2 (1:500, Abcam, Cambridge, MA), and anti-GAPDH (1:100,000; Abcam, Cambridge, MA) primary antibodies. The secondary antibodies used were anti-mouse IgG HRP (1:10,000; Cell Signaling, Danvers, MA) or anti-rabbit IgG HRP (1:5,000, Cell Signaling, Danvers, MA). The rabbit polyclonal anti-NFX1-123 antibody was generously provided by Dr. Ann Roman. Animals were immunized with peptide composed of amino acids 1102–1120 of NFX1-123. All films were scanned using Epson Perfection V700 and imported using Adobe Photoshop.
6
Western Blot Analysis of Cellular Fractions
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Cells were suspended in Radio Immunoprecipitation Assay lysis buffer (Thermo Scientific, PA, USA) supplemented with protease inhibitor (Sigma Aldrich). Cytosol and nuclear fractions were isolated according to manufacturer’s instruction (Thermo). Equivalent proteins were subjected to SDS-PAGE and transferred to polyvinyl difluoride membranes (Millipore Corp., Atlanta, GA, USA) for western blotting. After blocking membrane with 5% non-fat dry milk for 2 h, we incubated it with primary antibodies at 4°C overnight. Primary antibodies included anti-FLAG M2 (Sigma), anti-Omp25, anti-Omp31 and anti-β-actin (Wuhan boster Biotech), anti-phospho-IκB, anti-IκB, anti-p65, anti-TRAF6, anti-IRAK1, anti-IRAK2, and anti-Histone H3 (Cell Signaling Technology). HRP-conjugated secondary antibodies were incubated at room temperature for 1 h. ECL (Bio-Rad) was used for enhanced chemiluminescence detection according to the manufacturer’s instructions.
7
Molecular Interactions in TAK1 Signaling
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Anti-phospho-TAK1 (Thr-187), rabbit anti-TAK1, anti-TRAF2, anti-TRAF6, anti-TAB1, anti-TAB2, anti-phospho-IKKα/β (Ser176/180), anti-phospho-MKK7 (Ser-271/Thr-275), and anti-MKK7 antibodies were purchased from Cell Signaling Technology. Anti-TAB3 antibody was from Abcam. Anti-Myc antibody, normal mouse IgG and normal rabbit IgG were from Millipore. Anti-Flag antibody (M2) was obtained from Sigma. Anti-HA, anti-α-Tubulin, and horseradish peroxidase-conjugated secondary antibodies were from Santa Cruz Biotechnology. Rabbit anti-Vpr antibody was provided by National Institutes of Health (NIH) AIDS Research and Reference Reagent Program. Mouse anti-p24 and anti-TAK1 were generated by immunizing mice with the corresponding full length proteins purified form E. coli BL21 (DE3). 4′, 6-diamidino-2-phenylindole (DAPI) and PMA were purchased from Sigma. Fluorescein-conjugated anti-mouse and anti-rabbit secondary antibodies were purchased from Jackson ImmunoResearch Laboratories. Serine/Threonine phosphatase inhibitor Calyculin A was purchased from Cell Signaling Technology.
8
Protein Interaction Analysis of LACC1
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Western blot was performed using anti-LACC1 antibodies (Abcam, diluted 1:1,000). Loading controls included GAPDH (EMD Millipore, clone 6C5, diluted 1:10,000) for cell lysates, α-tubulin (Cell Signaling, diluted 1:1,000) for cytoplasmic protein and MTCO2 (Abcam, clone MTC02, diluted 1:1,000) for mitochondrial proteins. Mitochondrial and cytoplasmic subcellular fractionation was conducted using the Mitochondria isolation kit (ab 110170, Abcam). LACC1 (with anti-LACC1, Santa Cruz Biotechnology, using 4 μg antibody), RIP2 (with anti-RIP2, BD Biosciences, clone 25/RIG-G, using 4 μg antibody) or NOD2 (with anti-NOD2, Cayman Chemical, using 4 μg antibody) were immunoprecipitated from transfected HEK293 cells or MDMs with antibody-bound protein A sepharose beads. Associated proteins were examined with anti-SDHA (Abcam, clone 2E3GC12FB2A[E2], diluted 1:500), anti-LACC1 (Santa Cruz Biotechnology, diluted 1:1,000), anti-IRAK1 (clone D51G7, diluted 1:1,000), anti-TRAF6 (clone D21G3, diluted 1:1,000), anti-phospho-ERK (diluted 1:1,000), anti-phospho-p38 (clone 28B10, diluted 1:1,000) or anti-phospho-IκBα (clone 5A5, diluted 1:1,000; all Cell Signaling Technology) antibodies.
9
Antibody Validation for Signaling Pathway
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Specific anti-HA, anti-Flag anti-Myc, anti-TRAF6, anti-pho-TAK1, anti-TAK1, anti-pho-IKKβ, anti-IKKαβ, and anti-GAPDH antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). The anti-p62 and anti-ECSIT antibodies were purchased from Abcam (Cambridge, MA, USA). Mouse TrueBlot ULTRA: anti-Mouse Ig HRP was purchased from Rockland Immunochemicals, Inc. (Limerick, PA, USA).
10
TLR-Mediated Signaling Pathway Analysis
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Lipopolysaccharide (LPS, E. coli 0111: B4, TLR4 ligand), poly(I:C) (TLR3 ligand), and CpG ODN (TLR9 ligand) were purchased from Sigma-Aldrich. ChIP Grade Protein G Magnetic Beads, Cell Lysis Buffer, Anti-Myd88, Anti-Traf6, Anti-p65, Anti-p-p65 (Ser536), Anti-Ikkα/β, Anti-p-Ikkα/β (S176/180), Anti-IκBα, Anti-p-IκBα (S32), Anti-ERK, Anti-p-ERK (Thr202-Tyr204), Anti-JNK, Anti-p-JNK (Thr183-Tyr185), Anti-p38, Anti-p-p38 (Thr180-Tyr182) and Anti-Syk and Anti-p-Syk (Tyr-525/526) were from Cell Signaling Technology. Anti-DAP12 was purchased from Abcam. Anti-β-actin, Anti-rabbit IgG-HRP, and anti-mouse IgG-HRP were from Santa Cruz Biotechnology. Anti-LaminA/C, Anti-Flag and Anti-Myc and Anti-V5 were purchased from Proteintech. Anti-Ocilrp2 (AF3370) was from purchased R&D. The NF-κB inhibitor BAY11-7082 (10 μM), Tbk inhibitor Amlexanox (10 μM), Mek inhibitor PD98059 (10 μM), PI3K inhibitor Wortmannin (5 μM), Erk inhibitor SHC772984 (10 μM), Jnk inhibitor SP600125 (10 μM), or p38 inhibitor SB203580 (10 μM), Syk inhibitor R406 (5 μM) were from Selleckchem.
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