Cell proteins were extracted with RIPA lysis buffer (Beyotime, Beijing, China). The protein concentrations were determined by bicinchoninic acid (BCA) protein assay kit (Cwbio, Jiangsu, China), and then boiled at 95°C with 6 × loading buffer for 8 min. Samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immediately transferred onto 0.22 μm polyvinylidene fluoride membranes (Millipore, USA), the membranes were blocked with Tris-buffered saline with Tween 20 (TBST) containing 5% nonfat milk at room temperature for 60 min. After washing three times, the membranes were incubated overnight at 4°C with anti-TLR4 (1:1,000 dilution, AF7017, Affinity). Then blots were bound with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:20,000 dilution, Proteintech) for 60 min at 37°C. After washing again with TBST three times, experimental results were obtained by the pierce enhanced chemiluminiscent (ECL) western blotting kit (Thermo Scientific), and imaged by chemiluminescence system (BIO-RAD, USA).
Anti tlr4
Manufactured by Affinity Biosciences
Sourced in United States
Anti-TLR4 is a laboratory reagent that binds and neutralizes the Toll-like receptor 4 (TLR4) protein. TLR4 is a key component of the innate immune system that recognizes and responds to bacterial lipopolysaccharides. Anti-TLR4 can be used in research applications to study the role of TLR4 in various biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Af7017, by Affinity Biosciences (2 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
0.22 μm polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Proteintech (1 mentions)
Bca protein assay kit, by CWBIO (1 mentions)
Anti cd45, by Cell Signaling Technology (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Anti sqstm1 p62, by Cell Signaling Technology (1 mentions)
Anti ulk1, by Cell Signaling Technology (1 mentions)
Pierce classic magnetic ip co ip kit, by Thermo Fisher Scientific (1 mentions)
Ab32419, by Abcam (1 mentions)
Ab207612, by Abcam (1 mentions)
Anti glyceraldehyde 3 phosphate dehydrogenase gapdh, by Cell Signaling Technology (1 mentions)
Anti phospho ulk1 ser757, by Cell Signaling Technology (1 mentions)
Ass1009, by Abcepta (1 mentions)
Af3308, by Affinity Biosciences (1 mentions)
Anti mtor, by Affinity Biosciences (1 mentions)
Anti atg5, by Abways (1 mentions)
Anti integrin α5, by Abways (1 mentions)
Anti rab7, by Cell Signaling Technology (1 mentions)
5 protocols using anti tlr4
1
Western Blot Analysis of TLR4 Protein
2
Immunohistochemical Analysis of Liver Tissue
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Immunohistochemical analyses of the healthy/injured liver were performed on 5-μm-thick tissue sections that underwent antigen retrieval using citrate buffer solution. The sections were blocked using 5% bovine serum albumin solution. Following this, the primary antibodies, which included anti-CD45 (Cell Signaling Technology) and anti-TLR4 (Affinity Biosciences LTD.) were incubated with the tissue sections overnight at 4°C. The sections were then incubated with Horseradish Peroxidase (HRP) secondary antibodies (Proteintech, China) according to the corresponding sources. The sections were developed using a diaminobenzidine substrate kit and counterstained using haematoxylin. Tissue images were obtained using a microscope.
3
Western Blot Antibody Characterization
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For Western blot analysis, the following antibodies were used: anti-LC3B (2775; Cell Signaling Technology), anti-SQSTM1/P62 (5114; Cell Signaling Technology), anti-ATG7 (DF6130; Affinity Biosciences), anti-ATG5 (CY5766; Abways), anti-fibronectin (ab32419; Abcam), anti-integrin α5 (CY5979; Abways), anti-integrin β1 (CY5469; Abways), anti-phospho-Beclin-1 (Ser15) (84966; Cell Signaling Technology), anti-BECN1 (ab207612; Abcam), anti-TLR2 (AF7002; Affinity Biosciences), anti-TLR4 (AF7017; Affinity Biosciences), anti-CST (AB0055; Abways), anti-Rab7 (9367; Cell Signaling Technology), anti-phospho-MTOR (Ser2448) (AF3308; Affinity Biosciences), anti-MTOR (AF6308; Affinity Biosciences), anti-phospho-ULK1 (Ser757) (6888; Cell Signaling Technology), anti-ULK1 (8054; Cell Signaling Technology), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (5174; Cell Signaling Technology). Horseradish peroxidase-labeled goat anti-rabbit (ASS1009; Abgent) secondary antibodies were used. The transfection reagents were Lipofectamine 2000 (11688-019; Invitrogen) and HiPerFect (Y5-301705; Qiagen). The immunoprecipitation (IP) reagent was included in the Pierce classic magnetic IP/co-IP kit (88804; Thermo Scientific).
4
Mitochondrial Dynamics Regulation Assays
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Mitochondrial membrane potential assay kit (C3601), Fast silver staining kit (P0017S), and cell mitochondria isolation kit (C2006) were purchased from Beyotime (Shanghai, China). Reactive oxygen species (ROS) assay kit (KGT010-1) and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (KGA312) were purchased from KeyGen Biotech (Nanjing, China). Duolink In Situ Probemaker (DUO92010/DUO92009) and Duolink flowPLA detection kit-Red (DUO94001) were purchased from Sigma-Aldrich (Saint Louis, USA). All siRNA and shRNA were purchased from Origene (Beijing, China). Anti-OPA1 [DF8587, 1:500 for Western blot (WB), 1/100 for immunofluorescence (IF), and 1:200 for immunoprecipitation (IP)], anti-TRAF3 (DF7181, 1:1000 for WB, 1:100 for IF, and 1:200 for IP), anti-MFN1 (DF7543, 1:1000 for WB, 1:100 for IF, and 1:200 for IP), anti-TLR4 (AF7017, 1:1000 for WB, 1:100 for IF, and 1:200 for IP) monoclonal antibodies and goat anti-rabbit/mouse-horseradish peroxidase-conjugated secondary antibody (S0001/S0002, 1:5000 dilution) were purchased from Affinity (Cincinnati, USA).
5
Western Blot Analysis of Nampt and TLR4 Expression
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Tissue samples were homogenized using liquid nitrogen and lysed in RIPA lysis buffer (Applygen Technologies Inc.) for 30 min. Lysates were then centrifuged at 4˚C for 10 min at 12,000 x g, and the supernatants were collected. Total proteins were separated by SDS-polyacrylamide gel electrophoresis, and transferred onto PVDF membranes (Millipore). The membranes were incubated with the appropriate primary antibodies, including rabbit polyclonal anti-Nampt (cat. no. DF6059; Affinity Biosciences; dilution, 1:500), rabbit polyclonal anti-TLR4 (cat. no. bs-20594R; Beijing Biosynthesis Biotechnology Co., Ltd.; dilution, 1:1,000) and mouse monoclonal anti-GAPDH (cat. no. TA-08; ZSBIO; dilution, 1:2,000) at 4˚C overnight, followed by incubation with HRP-conjugated goat anti-rabbit IgG (H+L) (cat. no. ZB-2301; ZSBIO; dilution, 1:2,000) or HRP-conjugated goat anti-mouse IgG (H+L) (cat. no. ZB-2305; ZSBIO; dilution, 1:2,000) at 4˚C for 2 h. Signals were visualized with the SuperSignal® West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Inc.), and the band density was quantified using Quantity One software (version 4.6.9; Bio-Rad Laboratories, Inc.).
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