Total protein was extracted from intestinal tissue and Caco-2 cells using a commercial protein isolation kit (KeyGEN Biotech, Nanjing, China). Equal protein amounts from the samples were analyzed using 10%-15% SDS-PAGE (Bio-Rad, Hercules, CA, United States) and then transferred to PVDF membranes (Millipore, Bedford, MA, United States). The membranes were blocked with 5% nonfat milk or 5% BSA in TBS-Tween buffer (0.1% Tween-20; pH 7.5) for 1 h at 37 °C. After blocking, the membranes were incubated with primary antibodies against total DRP1, DRP1 Ser637 (Abcam, Cambridge, United Kingdom), PINK1 (Santa Cruz Biotechnology, Santa Cruz, CA, United States), caspase-3 (Proteintech, Wuhan, China), and β-actin (ZSGB-BIO, Beijing, China) overnight at 4 °C. After washing, the membranes were incubated with the corresponding homologous horseradish peroxide-conjugated secondary antibodies for two hours at 37 °C. The bands were visualized using enhanced Chemiluminescence Plus reagents (Beyotime Institute of Biotechnology, China). Spectrophotometric analysis was performed with a BioSpectrum-510 multispectral imaging system (UVP, Upland, CA, United States) and signals were analyzed using Gel-pro Analyzer Version 5.0 (Media Cybernetics, Rockville, MD, United States).
Chemiluminescence plus reagents
Manufactured by Beyotime
Sourced in China
Chemiluminescence Plus reagents are a set of chemical compounds used to facilitate chemiluminescence-based detection and analysis in various laboratory applications. The core function of these reagents is to generate luminescent signals that can be measured and quantified to detect and analyze target analytes.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Gel pro analyzer version 5, by Media Cybernetics (1 mentions)
Sds page, by Bio-Rad (1 mentions)
Pink1, by Santa Cruz Biotechnology (1 mentions)
Caspase 3, by Proteintech (1 mentions)
β actin, by ZSGB-BIO (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Anti p akt, by Cell Signaling Technology (1 mentions)
Anti p iκbα, by Cell Signaling Technology (1 mentions)
Anti β arrestin2, by Cell Signaling Technology (1 mentions)
Anti p nf κb p65, by Cell Signaling Technology (1 mentions)
Anti nf κb p65, by Cell Signaling Technology (1 mentions)
Dexamethasone dxm, by Merck Group (1 mentions)
Anti iκbα, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Proteintech (1 mentions)
Foxm1, by GeneTex (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Cyclin e, by Santa Cruz Biotechnology (1 mentions)
Gapdh, by Proteintech (1 mentions)
3 protocols using chemiluminescence plus reagents
1
Western Blot Analysis of Mitochondrial and Apoptotic Proteins
2
Alleviating Neuroinflammation through DRD2 Antagonism
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RoMS (Batch number: 20180902) were provided by Shandong Luye Pharmaceutical Co., Ltd. (Shandong, China). LPS (from Escherichia coli 011: B4, Catalog No: L2640) was purchased from Sigma Aldrich (St. Louis, MO, United States). Dexamethasone (DXM) (CAS No.: 50-02-2) was purchased from Sigma Aldrich (St. Louis, MO, United States). R121 (CAS No.:98,185-20-7), a specific DRD2 antagonist, was purchased from Sigma Aldrich (St. Louis, MO, United States). The ELISA kits of TNF-α and IL-6 were provided by Suolaibao, Inc. (Beijing, China). The biochemical kits of AST and ALT were purchased from Nanjing Jiancheng Co., Ltd. (Jiangsu, China). The primary antibodies used in this study are as follows: anti-TLR4 and anti-IL-6 (Santa Cruz Biotechnology, Dallas, TX, United States), anti-dopamine D2 receptor (Abcam, Cambridge, MA, United States), Anti-β-arrestin2, anti-p-Akt, anti-Akt, anti-p-IκBα, anti-IκBα, anti-p-NF-κBp65, anti-NF-κBp65 (Cell Signaling Technology, Danvers, MA, United States), anti-GAPDH (Proteintech Group, Inc., Wuhan, China). BCA protein assay kit and chemiluminescence plus reagents were purchased from Beyotime Biotechnology (Shanghai, China).
3
Western Blot Analysis of Cell Signaling
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The treated cells were collected and lysed with RIPA lysis buffer (Beyotime, Shanghai, China). The protein concentration was determined using BCA Protein Assay Kit (Beyotime, Shanghai, China). Equal amounts of proteins were separated by SDS‐PAGE and transferred to PVDF membranes (Millipore, MA, USA). After blocking, the membranes were immunoblotted with primary antibodies that were specific for ASPM, CyclinD1 and CyclinE (Santa Cruz, CA, USA); FoxM1 (GeneTex, CA, USA); P53, P21 and N‐cadherin (Cell Signaling Technology, MA, USA); GAPDH (Proteintech, Wuhan, China); and E‐cadherin, Vimentin, MMP1, MMP2, MMP9, RB and CDK2 (Wanlei Biotech, Shenyang, China). After washing with TBST, the membranes were incubated with appropriate secondary antibodies. The membranes were then exposed to enhanced Chemiluminescence‐Plus reagents (Beyotime, Shanghai, China). ChemiDoc™ XRS + chemiluminescence imaging system was used to capture the images. GAPDH protein was used as the internal reference to calculate the relative expression level of the protein by Image Lab 3.0 software.
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