Total proteins were extracted from the paw of CIA mice, and their concentration was determined using a BCA assay (Beyotime). Protein samples were applied and separated on 10% NuPAGE gel (Invitrogen), followed by transferring onto PVDF membranes (Millipore Inc.). Membranes were blocked in 5% non‐fat dry milk and 0.1% Tween‐20 for 1 h, followed by incubation overnight with primary antibody against mouse NF‐κB p50 (Abcam; ab32360), NF‐κB p65 (Cell Signalling; #8242T), Act 1 (Santa Cruz Biotechnology; sc‐100647), TRAF6 (Immunoway; YT4720), IL‐17RA (Absin Bioscience; abs140681) and GAPDH (Absin Bioscience; abs100005) diluted at 1:1000 in blocking solution. Next, the HRP‐conjugated secondary antibody (Absin Bioscience) at a dilution of 1:8000 was used to incubate the membranes for 2 h. Immunoreactive proteins were visualized using Tanon high‐sig ECL Western blotting Substrate and Tanon 5200 multifunction laser‐scanning system (Tanon).
Traf6
Manufactured by Immunoway
Sourced in United States
TRAF6 is a protein that plays a critical role in signal transduction pathways. It functions as an E3 ubiquitin ligase, which is involved in the regulation of various cellular processes, including immune response, inflammation, and cell survival.
Automatically generated - may contain errors
Lab products found in correlation
Ab32360, by Cell Signaling Technology (2 mentions)
Il 17ra, by Absin (2 mentions)
Hrp conjugated secondary antibody, by Absin (2 mentions)
Nf κb p65, by Cell Signaling Technology (2 mentions)
Bca assay, by Beyotime (2 mentions)
Gapdh, by Absin (2 mentions)
Nupage gel, by Thermo Fisher Scientific (1 mentions)
P nf κbp65, by Immunoway (1 mentions)
Hrp conjugated secondary antibody, by Abbkine (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
β actin, by Proteintech (1 mentions)
Proliferating cell nuclear antigen (pcna), by Immunoway (1 mentions)
Chemiluminescence system, by Beyotime (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Chemiluminescence kit, by Beyotime (1 mentions)
β actin, by Immunoway (1 mentions)
4 protocols using traf6
1
Quantifying Inflammatory Proteins in CIA Mice
2
Protein Expression Analysis in CIA Mice
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Total proteins were extracted from the paw of CIA mice and their concentration was determined using a BCA assay (Beyotime, Nanjing, CHN). Protein samples were applied and separated on 10% NuPAGE gel (Invitrogen, New York, NY), followed by transferring onto PVDF membranes (Millipore Inc., Darmstadt, Germany). Membranes were blocked in 5% non-fat dry milk and 0.1% Tween-20 for 1 h, followed by incubation overnight with primary antibody against mouse NF-κB p50 (Abcam, Cambridge, UK, ab32360), NF-κB p65 (Cell signaling, Danvers, MA, #8242T), Act 1 (Santa Cruz Biotechnology, Santa Cruz, CA, sc-100647), TRAF6 (Immunoway, Suzhou, CHN, YT4720), IL-17RA (Absin Bioscience, Shanghai, CHN, abs140681), and GAPDH (Absin Bioscience, Shanghai, CHN, abs100005) diluted at 1:1000 in blocking solution. Next, the HRP-conjugated secondary antibody (Absin Bioscience, Shanghai, CHN) at a dilution of 1:8,000 was used to incubate the membranes for 2 h. Immunoreactive proteins were visualized using Tanon high-sig ECL Western blotting Substrate and Tanon 5200 multifunction laser-scanning system (Tanon, CHN).
3
Western Blot Analysis of Cellular Proteins
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The cells were washed with PBS before being lysed in lysis buffer that can extract both cytoplasm proteins and nucleoproteins. The sample was stored at −80°C. Samples containing an equal amount of proteins were mixed with 5x SDS loading buffer and electrophoresed on a 10% SDS-PAGE gel. The proteins were then transferred onto PVDF membrane (Millipore). The membranes were blocked and probed with antibodies anti-NFAT2 (CST), TRAF6 (Immunoway), p-NF-κBp65 (Immunoway), PCNA (Immunoway), p65 (Immunoway), and β-actin (Proteintech group). The membranes were incubated with specific primary antibodies overnight at 4°C at a 1 : 1000 dilution. Subsequently, the membranes were washed with TBS/T for 15 min, three times, and incubated for 1 h with HRP-conjugated secondary antibodies (Abbkine). The protein was visualized with chemiluminescence, and a densitometric scanner was used to determine the density of the band. All experiments were repeated at least three times independently.
4
Western Blot Analysis of Liver Proteins
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Liver tissues were lysed in ice-cold RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) containing protease inhibitors and phosphatase inhibitors and then centrifuged at 12000 g for 15 min at 4°C and collected the supernatants carefully. Protein concentrations were determined by BCA protein assay kit (Beyotime Biotechnology, China). Equivalent amounts of protein were separated by 10% SDS polyacrylamide gel electrophoresis and transferred to PVDF membranes. The membranes were blocked in 5% (w/v) nonfat milk powder in TBST (Tris-buffered saline with 0.1% Tween) for 1 h at room temperature. The membranes were incubated with rabbit primary antibodies for TLR4, NF-κB, MYD88, TRAF6, P38, p-P38, JNK1/2, p-JNK1/2, and β-actin from ImmunoWay Biotechnology Company (Plano, TX, USA), respectively, diluted in blocking buffer at 4°C overnight. After washing with TBST for 3 times, the membranes were incubated with peroxidase-conjugated anti-rabbit secondary antibody (1 : 5000, Abcam USA) for 1 h at room temperature and then washed with TBST for 3 times. The immunoblots were developed using a chemiluminescence kit (Beyotime Biotechnology, China) and visualized by a chemiluminescence system (Tanon 5200). The band intensity was quantified using ImageJ, normalized to β-actin.
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