Western blot assay was carried out as previously described (Wang et al., 2019 (link)). The primary antibodies, including anti-CBS, anti-CSE, anti-3-MST, anti-cyclin E1, anti-cyclin D1, anti-cyclin-dependent kinase-2 (CDK2), anti-cyclin-dependent kinase-4 (CDK4), anti-p27, anti-p21, anti-beclin-1, anti-LC3A/B, anti-P62, anti-phosphatidylinositol 3-kinase (PI3K), anti-AKT, anti-mammalian target of rapamycin (mTOR), anti-phospho (p)-PI3K (Tyr199/Tyr458), anti-p-AKT (Ser473), and anti-p-mTOR (Ser2448) were purchased from Cell Signaling Technology (CST, Danvers, MA, United States). The primary antibodies, including anti-cleaved caspase (cas)-3, anti-cleaved cas-9, anti-cleaved poly adenosine diphosphate-ribose polymerase (PARP), anti-β-actin, and the horseradish peroxidase-conjugated secondary antibody were purchased from Proteintech (Chicago, IL, United States). The immunoreactive bands were visualized by a chemiluminescence detection system (Thermo Fisher, Waltham, MA, United States).
Anti cbs
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-CBS is a primary antibody product developed by Cell Signaling Technology. It is designed to detect cystathionine beta-synthase (CBS) protein in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Chemiluminescence detection system, by Thermo Fisher Scientific (1 mentions)
Anti p62, by Cell Signaling Technology (1 mentions)
Anti cse, by Cell Signaling Technology (1 mentions)
Anti 3 mst, by Cell Signaling Technology (1 mentions)
Anti p mtor ser2448, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Proteintech (1 mentions)
Anti cyclin d1, by Cell Signaling Technology (1 mentions)
Anti beclin 1, by Cell Signaling Technology (1 mentions)
Anti lc3a b, by Cell Signaling Technology (1 mentions)
Anti cyclin e1, by Cell Signaling Technology (1 mentions)
Anti p21, by Cell Signaling Technology (1 mentions)
Polyvinylidine difluoride membrane, by Merck Group (1 mentions)
Anti phospho ampkα, by Cell Signaling Technology (1 mentions)
Anti ampkα, by Cell Signaling Technology (1 mentions)
Anti rabbit 7074, by Cell Signaling Technology (1 mentions)
Anti tnf α, by Cell Signaling Technology (1 mentions)
Anti mouse 7076, by Cell Signaling Technology (1 mentions)
Anti pepck, by Santa Cruz Biotechnology (1 mentions)
Anti nf κb p65, by Cell Signaling Technology (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
4 protocols using anti cbs
1
Comprehensive Western Blot Analysis
2
Quantifying Hepatic CSE and CBS Levels
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To confirm the CSE and CBS protein expression levels, 10 μg protein samples lysed in RIPA buffer (25 mM Tris–HCl, pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS; Thermo Scientific) were prepared from the livers taken from each group. The proteins were resolved on a 10% gel and transferred to a polyvinylidine difluoride membrane (Millipore, Bedford, MA, USA). The membrane was then incubated overnight with primary antibodies at 4 °C. Anti-CSE (MBS2015730) was obtained from My BioSource (San Diego, CA, USA), and anti-CBS (#14782), anti-TNF-α (#6945), anti-IL-6 (#12153), anti-phospho-NF-κB p65 (#3033), anti-NF-κB p65 (#4764), anti-phospho-AMPKα (#2535), anti-AMPKα (#2532), and anti-β-actin (#4967) were obtained from Cell Signaling Technology (Canvers, MA, USA). Anti-PEPCK was purchased from Santa Cruz (Dallas, TX, USA). The secondary antibodies were incubated for 1 h at room temperature. Anti-Rabbit (#7074) and anti-Mouse (#7076) were obtained from Cell signaling Technology (Canvers, MA, USA). Bands were detected using an enhanced chemiluminescence system (Amersham, Buckinghamshire, UK).
3
Extraction and Western Blot Analysis of Pancreatic Proteins
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Pancreatic tissues were frozen at −80 °C until homogenization in extraction buffer (100 mg/ml) on ice. The extraction buffer contained 20 mM Tris–HCl (pH 7.5), 1 mM EDTA, 150 mM NaCl, 0.1% SDS, 1% Igepal CA-630, 30 mM sodium pyrophosphate, 50 mM sodium fluoride, 50 μM sodium orthovanadate (all from Sigma-Aldrich, St. Louis, MO, USA) and a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA) at a concentration of 0.4% (v/v). For western blotting under non-reducing conditions, 50 mM NEM was added to the extraction buffer. Protein extracts were added to reducing or non-reducing sample buffer and separated by SDS-PAGE. For electrophoresis under reducing conditions, the following sample buffer was used: 130 mM Tris–HCl, pH 6.8, 10% glycerol, 0.05% bromophenol blue, 2% SDS, and 100 mM DTT. When electrophoresis was performed under non-reducing conditions, the sample buffer had the same composition but without DTT. The following antibodies were used: anti-CBS (1:1000; Cell Signalling Technology, Danvers, MA, USA), anti-nitro-tyrosine (Cell Signaling Technology, Danvers, MA, USA) and anti-beta tubulin (1:1000, Abcam, Cambridge, UK).
4
Protein Expression Analysis in Cell Lysates
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Cells were seeded in 6-well plates at 105 cells/well. On the day of the experiment, cell medium was removed, cells were washed with PBS, and PathScan sandwich ELISA lysis buffer (Cell Signaling Technology, Danvers, MA, USA) supplemented with 1X Protease/Phosphatase Inhibitor Cocktail (Cell Signaling Technology) was added. After centrifugation, protein content of the lysates was quantified using the Bradford assay. For this, 20 µg of total protein was loaded onto 4–12% bis-tris gel and transferred to a nitrocellulose membrane with iBolt2 apparatus. Membranes were blocked for 1 h at room temperature and then incubated with specific primary antibodies overnight at 4 °C. The antibodies used in this study were: rabbit anti-SELENBP1 (Abcam, 1:1000), rabbit anti-Adiponectin (Sigma, 1:500), anti-CBS (Cell Signaling Technology, 1:1000), rabbit anti-CSE (Abcam 1:500), rabbit anti 3-MST (Abcam, 1:300), and mouse anti-β-Actin (Cell Signaling Technology, 1:5000). The latter was used as a loading control. Detection was performed with ECL Prime solution (Sigma-Aldrich, St Louis, MO, USA) using the Azure 600 analyzer (Azure biosystems).
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