The total proteins were extracted and separated by 10% or 15% SDS-PAGE and were transferred to polyvinylidene difluoride membranes (Millipore, Bedford, Massachusetts, USA) using a semi-dry Gel Transfer Device (Bio-Rad, Hercules, California, USA). The membranes were blocked using 5% non-fat milk and probed with primary antibodies and HRP-conjugated secondary antibodies. Antigen-antibody complexes were visualised using a chemiluminescent ECL detection system and analysed using a ChemDoc XRS+image analyser. GAPDH or β-actin was used as an internal control. The antibodies used included anti-β5i (1:5000, ab180606, Abcam), anti-RIG-I (1:2000,3743, Cell Signaling Technology), anti-Phospho-NF-κB p65 (1:1000, 3033, Cell Signaling Technology), anti-Phospho-IRF3 (1:500, ab76493, Abcam), anti- IFNβ (1:500, ab275880, Abcam), anti- MxA (1:500, sc-166412, Santa Cruz), anti-MuRF1 (1:2000, ab172479, Abcam), anti-β-actin (1:5000, YM3028, Immunoway) and anti-GAPDH (1:10000, YM3029, Immunoway). The selective β5i inhibitor PR-957 was purchased from Selleck Chemicals (Houston, Texas, USA).
Ym3028
Manufactured by Immunoway
Sourced in United States, China
The YM3028 is a versatile laboratory equipment designed for general research and analytical applications. It features precise temperature control and advanced data logging capabilities. Further details on the specific intended use of this product are not available.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Thermo Fisher Scientific (4 mentions)
Ym3029, by Immunoway (2 mentions)
Ym3408, by Immunoway (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Ab180606, by Abcam (1 mentions)
Ab172479, by Abcam (1 mentions)
Ab76493, by Abcam (1 mentions)
Gel transfer device, by Bio-Rad (1 mentions)
Anti rig 1, by Cell Signaling Technology (1 mentions)
Pr 957, by Selleck Chemicals (1 mentions)
Sc 166412, by Santa Cruz Biotechnology (1 mentions)
Anti phospho nf κb p65, by Cell Signaling Technology (1 mentions)
Ab124964, by Abcam (1 mentions)
Ab45688, by Abcam (1 mentions)
Ab260043, by Abcam (1 mentions)
Ab52915, by Abcam (1 mentions)
Protease and phosphatase inhibitor, by Selleck Chemicals (1 mentions)
Enhanced chemiluminescence detection kit, by Fdbio (1 mentions)
Bca protein assay kit, by Fdbio (1 mentions)
Ripa buffer, by Fdbio (1 mentions)
9 protocols using ym3028
1
Western Blot Analysis of Immune Signaling
2
Western Blot Analysis of Fibrosis Markers
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The expression levels of α-SMA, COL1α1, FN1, and vimentin were examined by WB. The following antibodies were used: anti-α-SMA (Abcam, ab124964), anti-COL1α1 (Abcam, ab260043), anti-FN1 (Abcam, ab45688), anti-vimentin (Abcam, ab92547) and anti-β-actin (Immunoway, YM3028). Details are provided in the Additional file 2 : Methods.
3
Protein Extraction and Western Blot Analysis
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Total protein was extracted using RIPA buffer (Fdbio science, Hangzhou, China) containing 1% protease and phosphatase inhibitors (Bimake, USA). Protein quantification was performed using BCA Protein Assay Kit (Fdbio science, Hangzhou, China). Protein samples of 40ug from each group were separated by 4-20% SDS-PAGE (GenScript, Nanjing, China) and then transferred to 0.22 μm PVDF membranes (Millipore, USA). Following a 2-hour blocking step in 3% Bovine Serum Albumin (BSA) at room temperature, the membranes were incubated overnight at 4℃ with primary antibodies including anti‐β-actin (1:5000; YM3028, Immunoway), anti-MMP3 (1:1000, ab52915, Abcam), anti‐E‐cadherin (1:500; YT1454, Immunoway), anti‐ZO-1 (1:500; YN1410, Immunoway), anti-N-cadherin (1:500, YT2988, Immunoway), anti-β-Catenin (1:1000, YM3403, Immunoway), anti-Snail (1:500, YT4351, Immunoway) and anti-Vimentin (1:500, YT4880, Immunoway). The next day, the membranes was washed 3 times with TBST and incubated with HRP-secondary antibody for 2 hours at room temperature. Immunoblots were detected with an imaging system (Bio-Rad, USA) and an enhanced chemiluminescence detection kit (Fdbio science, Hangzhou, China). GAPDH was selected as a loading control.
4
Western Blot Analysis of Inflammatory Proteins
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Protein (20–50 μg) was extracted from the ipsilateral spinal cord dorsal horn and separated with 10% SDS-PAGE gel, transferred onto a nitrocellulose membrane, and incubated with antibody against CXCR4 (1:1000; ab124824, Abcam), VDUP-1 (1:1000; sc-271238, Santa Cruz Biotechnology), NLRP3 (1:500; NBP1-77080, Novus Biologicals), apoptosis-associated speck-like molecule containing CARD domain (ASC; 1:1000; sc-22514-R, Santa Cruz Biotechnology), Caspase1 (1:1000; ab-1872, Abcam), Caspase1 p10 (1:1000; sc-514, Santa Cruz Biotechnology), and IL-1β (1:1000; ab9722, Abcam) or control β-Actin (1:5000; YM3028, ImmunoWay). Blots were washed and incubated in HRP-linked anti-rabbit IgG antibody (1:5000; 7074, Cell Signaling Technology) and HRP-linked anti-mouse IgG antibody (1:5000; 7076, Cell Signaling Technology). Protein blots were visualized using Clarity ECL Substrates (Biorad).
5
Western Blot Analysis of ADR-β2 Protein
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Total proteins were isolated from tissue samples in each group using RIPA buffer for Western blot (WB) assays. The extracted proteins (40 μg per sample as determined by the BCA protein assay) were subjected to 90 minutes of sodium dodecyl sulfate–12% polyacrylamide gel electrophoresis (SDS-PAGE) with β-actin as an internal reference (YM3028, Immunoway). The proteins were then transferred to polyvinylidene difluoride (PVDF) membranes and blocked for 2 hours with 5% nonfat milk in TBST. Monoclonal antibodies against ADR-β2 (dilution 1:5000 in 5% nonfat milk-TBST, Abcam) were added onto the membranes and incubated at room temperature for 10 minutes, then overnight at 4°C. After washing with TBST, the membranes were incubated for 40 minutes at room temperature with 1:2000 HRP-conjugated secondary antibody–labeled IgG antibody diluted in 5% nonfat milk-TBST. Enhanced chemiluminescence (ECL) detection reagent (Amersham Pharmacia Biotech, Amersham, United Kingdom) was added to the membranes for 3 minutes, and the images were exposed to radiographic film. Finally, film autoradiograms were analyzed and quantified using the Quantity One 4. 60 software.
6
Western Blot Analysis of Liver Proteins
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Total protein was extracted from the liver tissue and separated by denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were then transferred onto polyvinylidene difluoride membranes (MilliporeSigma, Burlington, MA, USA). The membranes were blocked with 5% milk for 1 h and incubated overnight at 4 °C with primary antibodies against TLR4 (Proteintech Group, Rosemont, IL, USA; 19811-1-AP), MyD88 (Proteintech Group, 23230-1-AP), NF-κB p65 (Immunoway, Plano, TX, USA; YM3111), phospho-NF-κB p65 (Immunoway, YP0847), and β-actin (Immunoway, YM3028). The membranes were washed three times with phosphate-buffered saline with Tween-20 and incubated with secondary antibodies for an hour. Goat anti-rabbit IgG-HRP (Immunoway, RS0002) and goat anti-mouse IgG-HRP (Immunoway, RS0001) were used as secondary antibodies. Dilutions for primary antibodies and secondary antibodies were 1:1000 and 1:10,000, respectively. Band intensities were quantified using the Image J software (version 1.52) and normalized to β-actin levels. Original blots can be found in the accompanying Source Data file.
7
Protein Expression Analysis of Harvested Lung Tissues
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Harvested lung tissues were immersed in RIPA lysis buffer (HEART, Xi'an, China), and the concentrations of protein were measured by BCA protein assay kit (Pierce, Rockford, IL, USA) according to the manufacturer's instructions. Equal amounts of protein were resolved and separated on 8%-15% SDS-PAGE and then transferred onto polyvinylidene di uoride (PVDF) membranes (Bio-Rad). Membranes were blocked with nonfat dry milk (5%, w/v) for 1h, and then incubated overnight at 4°C with the speci c primary antibodies against β-actin (YM3028, Immunoway, TX, USA, 1:1000 dilution), FOXM1 (no 32671, Signalway Antibody, Pearland, TX, 1:1,000 dilution), LC3B (18725-1-AP, Proteintech, Chicago, IL, USA, 1:800 dilution), FAK (12636-1-AP, Proteintech, Chicago, IL, USA, 1:800 dilution) and p-FAK (sc-374668; Santa Cruz, 1:600 dilution). After washing blots three times in PBST, membranes were soaked in horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG secondary antibody (AP156P or AP127P, Sigma-Aldrich, St. Louis, MO, USA, 1:5000) for 1 hours. Blots were visualized by the enhanced chemiluminescence detection system (Amersham Bioscience), and the band densities were measured using Quality One software (Bio-Rad).
8
Aortic Protein Expression Analysis
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Aortic proteins were lysed and extracted using RIPA buffer (89900, Thermo Scientific, USA) containing a protease and phosphatase inhibitor cocktail (78443, Thermo Scientific, USA). Proteins were separated by SDS-PAGE and transferred to PVDF membranes. The primary antibodies used for immunoblotting were PI3K (1:10,000, YM3408, Immunoway, China), AKT (1:4,000, 4691, CST, USA), p-AKT-T308 (1:1,000, 13038, CST, USA), p-AKT-S473 (1:1,000, 4060, CST, USA), P21 (1:1,000, YM3453, Immunoway, China), P27 (1:1,000, 3686, CST, USA), and β-Actin (1:5,000, YM3028, Immunoway, China). The immunoreactive bands were displayed using an electrochemiluminescence (ECL) kit (DW101-01, TransGen Biotech, China) and analyzed by ImageJ software (ver. 1.44, National Institutes of Health, USA).
9
Protein Expression and Immunoblotting Analysis
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Proteins were lysed and extracted by RIPA buffer (89900, Thermo Scientific, United States). Subsequently, proteins were separated by SDS-PAGE and transferred to PVDF membranes. The primary antibodies used for immunoblotting were PI3K (1:4,000, YM3408, Immunoway, China), AKT (1:4,000, 4691, CST, United States), p-AKT (S473, 1:4,000, YP0006, Immunoway, China), P53 (1:4,000, Ab26, Abcam, China), P21 (1:2,000, ab109199, Abcam, China), P16INK4a (1:5,000, ab108349, Abcam, China), GAPDH (1:5,000, YM3029, Immunoway, China), and β-Actin (1:5,000, YM3028, Immunoway, China). The immunoreactive bands were visualized via electrochemiluminescence (ECL, DW101-01, TransGen Biotech, China) method and analyzed by ImageJ software (ver. 1.44, National Institutes of Health, United States).
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