The frozen C3–6 DRG samples of 25 rats were prepared after tissue lysis and centrifugation for SDS-polyacrylamide gel electrophoresis. The protein with SDS sample buffer was mixed with β-mercaptoethanol, separated under reducing conditions on a 5% SDS polyacrylamide gel, and finally transferred to a nitrocellulose membrane. Blocking was carried out in 3% BSA (VWR International LLC, Solon, OH, USA) solution for 30 minutes at room temperature. The membrane was incubated with rabbit anti-GABAAα2R (1:1,000, AGA-002; Alomone Labs) and rabbit anti-GAB-ABR1 (1:1,000, ab90883; Abcam). GAPDH (1:20,000; YM3029, ImmunoWay, Plano, TX, USA) was used as a standard. After overnight agitation at 4°C, the sample was then incubated with HRP-conjugated goat anti-rabbit antibody (1:10,000; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 1 hour at room temperature. The bands were acquired by enhanced chemiluminescence (GE Healthcare Biosciences, Piscataway, NJ, USA), and the blots were scanned and quantified using TotalLab Quant analysis software (TotalLab Limited, Newcastle Upon Tyne, England) and the result was expressed as the ratio of target gene immunoreactivity to GAPDH immunoreactivity.
Ym3029
Manufactured by Immunoway
Sourced in United States
The YM3029 is a laboratory equipment product designed for general scientific applications. It serves as a tool for various experimental and analytical tasks in research and testing environments. The core function of the YM3029 is to perform its intended tasks, without further interpretation or extrapolation on its usage.
Automatically generated - may contain errors
Lab products found in correlation
Ym3030, by Immunoway (3 mentions)
Ym3028, by Immunoway (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Enhanced chemiluminescence, by GE Healthcare (1 mentions)
Bovine serum albumin (bsa), by Avantor (1 mentions)
Hrp conjugated goat anti rabbit antibody, by Jackson ImmunoResearch (1 mentions)
P jak2, by Cell Signaling Technology (1 mentions)
P stat6, by Immunoway (1 mentions)
Bcl 2, by Immunoway (1 mentions)
Bcl2 associated x (bax), by Immunoway (1 mentions)
Rs23710, by Immunoway (1 mentions)
Rs23920, by Immunoway (1 mentions)
Odyssey infrared imaging system 3, by LI COR (1 mentions)
Gapdh, by Immunoway (1 mentions)
Ab180606, by Abcam (1 mentions)
Ab172479, by Abcam (1 mentions)
Ab76493, by Abcam (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Gel transfer device, by Bio-Rad (1 mentions)
Anti rig 1, by Cell Signaling Technology (1 mentions)
12 protocols using ym3029
1
Western Blot Analysis of GABA Receptors
2
Western Blot Analysis of JAK2/STAT6 Signaling
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The penumbra tissue was treated with RIPA lysis buffer (Yamei, Suzhou), and the protein concentration was measured. 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate the target proteins, which were then transferred to a nitrocellulose membrane. The membrane was blocked with 5% BSA for 1 h and then incubated with 5% BSA containing corresponding primary antibodies, including JAK2 (YT2426, Immunoway, Plano, TX, USA) (1 : 400), p-JAK2 (3776, Cell Signaling Technology, Danvers, MA, USA) (1 : 500), STAT6 (YT4454, Immunoway) (1 : 400), p-STAT6 (YP0256, Immunoway) (1 : 400), Bcl-2 (YT0470, Immunoway) (1 : 500), Bax (YT0455, Immunoway) (1 : 500), and GAPDH (YM3029, Immunoway) (1 : 5,000) on a shaker at 4°C overnight. On the second day, Tris-buffered saline-tween (TBST) was used to rinse the membranes 3 times, which were then incubated with secondary antibodies (RS23710, RS23920, Immunoway) (1 : 10,000) for 1 h. The bands were observed using an Odyssey Infrared Imaging System 3.0.29 (LICOR, Nebraska, USA).
3
Western Blot Analysis of Immune Signaling
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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).
4
Quantitative Analysis of Hepatic and Intestinal Transporters
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The livers and jejunum tissue were homogenized in ice-cold radio immunoprecipitation assay (RIPA) buffer [50 mM Tris HCl, 1 mM EDTA, 150 mM NaCl, 0.1% sodium dodecyl sulfate, 0.5% sodium deoxycholate, 1.0% Nonidet P-40, and phenylmethane sulfonyl fluoride (PMSF), pH 8.0]. The homogenate was centrifuged at 12,000 rpm for 20 min at 4°C. The supernatants were collected for Western blot analysis, which was performed as described previously (Kong et al., 2015 (link)). The antibodies used in the present study included anti-Mrp-2 (1:500; sc-5770, Santa Cruz; California, USA), anti-glyceraldehyde 3-phosphate dehydrogenase (anti-GAPDH) (1:20,000; YM3029, Immunoway; Texas, USA), anti-UGT1A1 (1:4,000; ab194697, Abcam; Cambs, UK), anti-P glycoprotein (1:1,000; ab170904, Abcam; Cambs, UK), anti-CES2 (1:1,000; DF6433, Affinity; Cambs, UK), and anti-Bcrp (1:200; sc-69988, Santa Cruz; California, USA). The gray intensities of the bands were quantified using ImageJ software (National Institute of Health, MD, USA).
5
Western Blot Analysis of TGF-β Signaling
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Whole cell lysates and western blotting were performed as described previously 9 (link). Antibodies against CD105 (ab169545, 1:1000), Smad2 (ab40855, 1:2000), pSmad3 (ab52903, 1:1000) were purchased from Abcam. An antibody against BMP9 (sc514211, 1:500) was purchased from Santa Cruz Biotechnology (USA). Antibodies against Smad1 (6944, 1:1000), pSmad1/5/8 (13820, 1:1000), and pSmad2 (3108, 1:1000) were purchased from Cell Signaling Technology. Antibodies against Smad3 (YM3417, 1:2000), GAPDH (YM3029, 1:20000), and β-tubulin (YM3030, 1:5000) were purchased from ImmunoWay Biotechnology.
6
Western Blot Analysis of Protein Expression
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Western blotting analysis was used to analyze the expression of proteins. Briefly, cell lysis was performed using radio immunoprecipitation assay lysis buffer containing protease and phosphatase inhibitor cocktails, and the protein concentrations were determined using a BCA assay. Subsequently, proteins (50 μg) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis for separation, after which they were electro-transferred onto a polyvinylidene fluoride membrane. After blocking with 5% BSA for 3 h at room temperature, the membranes were incubated with primary antibodies (CXCL12, Proteintech, 17402-1-AP; β-tubulin, Immunoway, YM3030; GAPDH, Immunoway, YM3029) at 4°C overnight followed by incubation with secondary antibodies (HRP-conjugated Affinipure Goat Anti-Rat IgG (H + L), Proteintech, and SA00001-15) for 2 h. Immunoreactive bands were visualized using a chemiluminescence kit, and the density of the bands was determined using scanning densitometry (Bio-Rad, Hercules, CA, USA).
7
Protein Extraction and Western Blot Analysis
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Protein was extracted in RIPA buffer, and the protein concentration was detected by the BCA kit (Beyotime P0011, China). After gel electrophoresis, PVDF membranes were incubated with primary and secondary antibodies, and the primary antibodies included anti-aP2 (1:500; 2H3-1G10; Novusbio, USA), anti-MMP-7 (1:500; abs146189; Absin, China), anti-MMP-9 (1:1000; ab76003; Absin), anti-MMP-12 (1:1000, ab52897; Absin), anti-CPT1 (1:1000; #97361; Cell Signaling Technology), anti-CD36 (1:1000; ab252922; Abcam), anti-P62 (1:1000; ab109012; Abcam), anti-LC3A/B (1:1500; #12741; Cell Signaling Technology), and GAPDH (1:5000; YM3029; Immunoway, USA). Images were processed by ECL chemiluminescence.
8
Western Blot Analysis of PDK4 Protein
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Cells were lysed in RIPA buffer (Beyotime, Shanghai, China) with 1% phenylmethylsulfonyl and then denatured. Protein concentrations were quantified by BCA assay. The lysate was separated via 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the proteins obtained were transferred to polyvinylidene difluoride (PVDF) membranes which were blocked with tris-buffered saline and 0.1% Tween 20 (TBST) containing 5% non-fat milk for 1 h at 37 °C and subsequently incubated with the following primary antibodies at 4 °C overnight: mouse anti-PDK4 monoclonal antibody (1:1000; ab110336, Abcam); mouse anti-GAPDH (1:1000; YM3029, Immunoway). The next day, the PVDF membranes were washed three times with TBST buffer and incubated with horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibody (1:3000; SA00001-1, Proteintech) for 1 h at room temperature. Signals were visualized using an enhanced chemiluminescence reagent (ThermoFisher, Waltham, MA, USA) according to the manufacturer’s instructions. The relative protein expression levels were normalized with GAPDH as the standard reference.
9
Quantitative Western Blot Analysis
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After lysed, the protein concentration was quantified using a bicinchoninic acid protein assay kit (Thermo Scientific). 30 μg protein per lane were loaded on SDS-PAGE gels. After electrophoresis, the proteins were transferred to PVDF membranes. The membranes were blocked with 5% BSA and incubated at 4°C overnight with the appropriate primary antibodies: IbA1(17198S, CST, 1:1,000), Nlrp3 (ab270449, Abcam, 1:1,000), caspase1 (ab179515, Abcam, 1:1,000), caspase1 (p20) (AG-20B-0042-C100, AdipoGen, 1:1,000), Il-1β (31202S, CST, 1:1,000), Gapdh (YM3029, Immunoway,1:5,000), and incubated with horseradish peroxidase-conjugated secondary antibodies (ASR1937 and ASR1651, Abcepta, 1:20,000). Protein bands were visualized by enhanced chemiluminescence (Bio-Rad, Hercules, CA, USA) and images were captured using a ChemiDoc™ MP imaging system (Bio-Rad). All samples were run in parallel with four replicates.
10
Comprehensive Immunoblotting Techniques
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Standard techniques were used for protein quantification, separation, transfer, and blotting in KLE and HEC-1A cells. Primary antibodies immunoblotting antibodies were used: ERRα (1: 500, ab76228, Abcam, London, UK), ERRα (1:500, E1G1J, Cell Signaling Technology, Massachusetts, USA), NLRP3 (1:500; 19,771-1AP, Proteintech, Wuhan, China), HIF-1α (1:500; 20,960-1AP, Proteintech, Wuhan, China), cleaved GSDMD (1:500; #36,425, Cell Signaling Technology, Massachusetts, USA), GSDMD (1:500, E9S1X, Cell Signaling Technology, Massachusetts, USA), GSDME (1:500, 84005S, Cell Signaling Technology, Massachusetts, USA), Caspase1 (22,915–1-AP, Proteintech, Wuhan, China), Caspase-1 (YT5743, Immunoway, USA), Caspase-1 (1:500, D7F10, Cell Signaling Technology, Massachusetts, USA), Caspase-3 (1:500, D3R6Y, Cell Signaling Technology, Massachusetts, USA), IL-18 (1:500,10,663–1-AP, Proteintech, Wuhan, China), cleaved-IL-1β (1:500, D3A3Z, Cell Signaling Technology, Massachusetts, USA), β-actin (1:2000, YM3028l, Immunoway, USA), β-tublin (1:2000, YM3030, Immunoway, USA), and GAPDH (1:2000; YM3029, Immunoway, USA).
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