Whole cell lysates from TcREG were prepared according to manufacturer’s instructions (#9803, Cell Signaling, Danvers, MA, USA). Proteins were resolved on NuPAGE 4–12% Bis-Tris gel (Life Technologies, Carlsbad, CA, USA), transferred on a nitro-cellulose membrane and blocked in Tris-Buffered Saline (TBS) containing 5% bovine serum albumin (BSA) and 0.1% Tween-20. The membranes were probed overnight with primary antibody against TRAF-6 (Millipore) and β-actin (Cell Signaling). Membranes were washed and incubated for 1 hour with horseradish peroxidase (HRP) labeled secondary antibody (Cell Signaling).
Traf6
Manufactured by Merck Group
Sourced in United States
TRAF6 is a laboratory equipment product manufactured by Merck Group. It is a recombinant protein that functions as an E3 ubiquitin ligase, playing a crucial role in various cellular signaling pathways.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh, by Merck Group (2 mentions)
Horseradish peroxidase hrp labeled secondary antibody, by Cell Signaling Technology (1 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Egfr 1005, by Santa Cruz Biotechnology (1 mentions)
Vegfa a 20, by Santa Cruz Biotechnology (1 mentions)
α tubulin, by Cell Signaling Technology (1 mentions)
P27kip1 c 19, by Santa Cruz Biotechnology (1 mentions)
Gapdh 14c10, by Cell Signaling Technology (1 mentions)
Nuclear extract kit, by BestBio (1 mentions)
Nf κb p65, by Santa Cruz Biotechnology (1 mentions)
Myd88 d80f5, by Cell Signaling Technology (1 mentions)
Iκbα c 21, by Santa Cruz Biotechnology (1 mentions)
P nfκb p65 ser 276, by Santa Cruz Biotechnology (1 mentions)
Cell lysis buffer 10, by Cell Signaling Technology (1 mentions)
Rt pcr kit, by Toyobo (1 mentions)
Myd88, by Merck Group (1 mentions)
P iκbα, by Merck Group (1 mentions)
Nf κb p65, by Merck Group (1 mentions)
Keap1, by Merck Group (1 mentions)
6 protocols using traf6
1
Western Blot Analysis of TRAF-6
2
Western Blot Analysis of Protein Targets
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Cells harvested at different time points were lysed and total protein extracts were used for western blotting using antibodies specific for AR (US Biological, Salem, MA), PSA (Santa Cruz Biotechnology, Dallas, TX), Cbl (C-15) (Santa Cruz Biotechnology, Dallas, TX), TRAF6 (Millipore, Temecula, CA), p27Kip1 (C-19) (Santa Cruz, Biotechnology), IRAK1 (F-4) (Santa Cruz, Biotechnology), ZFAND1 (A-14) (Santa Cruz Biotechnology), FGD4 (Epitomics, Burlingame, CA), ABHD3 (Biorbyt, Cambrige, UK), DOK4 (C-16) (Santa Cruz Biotechnology), EGFR (1005) (Santa Cruz Biotechnology), VEGFA (A-20) (Santa Cruz Biotechnology), α-tubulin (Cell Signaling, Danvers, MA), GAPDH (Sigma-Aldrich, St. Louis, MO). Total extracts (30–50 μg) were directly mixed with Lammeli sample buffer and separated on SDS-PAGE. Immunoblotting was performed using appropriate primary and horseradish peroxidase conjugated respective secondary antibodies. Positive signals were detected using a chemiluminiscence ECL kit (Pierce, Rockford, IL).
3
Western Blot Analysis of Lung Proteins
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Lung protein extracts were prepared using the Nuclear Extract Kit (BestBio, China) following the manufacturer's instruction. Equal amounts of protein were separated by SDS-PAGE and subsequently blotted on polyvinylidene fluoride membranes (220 mA, 65 min). Blots were blocked in TBS solution containing 0.1% Tween 20 and 5% nonfat dry milk overnight at 4°C. The following antibodies and dilutions were used: TLR7 (Catalog number 2633, Cell Signaling Technology; 1 : 1000); MyD88 (D80F5) (Catalog number 4283, Cell Signaling Technology; 1 : 1000); TRAF6 (Catalog number 04-451, Millipore, USA; 1 : 1000); IκB-α (c-21) (sc-371, Santa Cruz Biotechnology, 1 : 200); p-IKKα/β (Ser 176) (sc-21661, Santa Cruz Biotechnology, 1 : 200); NF-κB p65 (sc-109, Santa Cruz Biotechnology, 1 : 200); p-NF-κB p65 (Ser 276) (sc-101749, Santa Cruz Biotechnology, 1 : 200); and GAPDH (14C10) (Catalog number 2118, Cell Signaling Technology; 1 : 1000). The membranes were blotted with appropriate secondary antibodies (Immunology Consultants Laboratory, Inc., USA; 1 : 5000), and the blotted proteins were visualized by enhanced chemiluminescence using a commercially available kit (Millipore, USA).
4
Molecular Mechanisms of Oxidative Stress
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Antibodies against KDR, CD29, CD90, CD44, CD105, CD34, Von Willenbrand factor (vWF), GAPDH, NOX1, NOX4, HO-1, TLR4, MyD88, TRAF6, NFκB P65, p-IκBα, Keap-1, Nrf2 and 2′,7′-dichlorofluorescein diacetate (DCF-DA) were obtained from Sigma (St Louis, MO, USA). DMEM (high glucose), serum-free EBM, and fetal bovine serum (FBS) were from Nego (Shanghai, China). Cell lysis buffer (10 ×) was obtained from Cell Signaling Technology (CST, Boston, USA). The RT-PCR kit was purchased from TOYOBO (Shanghai, China). Other reagents included DAPI (Roche, Basel, Germany), hematoxylin and eosin (H&E; Toronto Chemicals, Toronto, ON, Canada), and trypsin (Sigma). All pairs of real-time PCR primers were synthesized by Shenggong Biotechnology (Shanghai, China). Other chemicals and reagents were of analytical grade.
5
Immunohistochemical Analysis of STX2 and TRAF6
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Paraffin-embedded specimens were cut into 4-μm sections and baked at 60 °C for 2 h. Immunohistochemistry was performed via SP-9000 detection kits that were purchased from ZSGB-BIO (Beijing, China). The slides were incubated overnight at 4 °C with a primary antibody, namely, rabbit polyclonal anti-STX2 (1:200, Proteintech, USA), or mouse polyclonal anti-tumor necrosis factor receptor-associated factor (TRAF6) (1:200, Sigma, USA). As the negative control, PBS was used in place of the primary antibody. The sections were incubated with 3,3-diaminobenzidine (DAB) for 1 min and counterstained with Hematoxylin and dehydrated with a gradient alcohol series and xylenes. The slides were sealed with neutral balsam. The sections were evaluated and scored independently by two researchers, who were blind to the patient outcomes, based on both the proportion of positively stained tumor cells and the intensity of the staining51 (link).
6
Immunofluorescence Staining of Cells
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The cells were seeded on coverslips at a density of 5 × 104 per well for 48 h and then were incubated with a primary antibody STX2 (1:100, Proteintech, USA) or TRAF6 (1:100, Sigma, USA). The coverslips were then incubated with rhodamine-conjugated or fluoresceinisothiocyanate (FITC)-conjugated goat antibodies against rabbit or mouse IgG (anti-rabbit IgG, Abcam; anti-mouse IgG, Abcam). After counterstaining with 4,6-diamidino-2-phenylindole (DAPI; Sigma), images were taken using an Olympus FV1000 confocal laser-scanning microscope.
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