We used RIPA buffer containing PMSF (Sigma, USA) and phosphorylase inhibitors (Sigma, USA) to lyse the cells and tissues. Cell extracts were centrifuged at 12000×g for 10 minutes at 4 °C. Firstly, protein specimens were separated based on their molecular weight by SDS-PAGE. Then, target proteins were transferred to PVDF membranes (Millipore, USA). Then, the PVDF membranes were incubated with the respective primary antibodies (Table S3 ) for overnight at 4 °C. This was followed by incubation with the secondary antibody (CST, USA) for 1-hour the subsequent day. The protein bands were visualized using ECL (Pierce, USA), and collected by ChemiDocTm XRS Molecular Imager system (Bio-Rad, USA). Finally, the Image J software was used to analyze the band densities.
Phosphorylase inhibitor
Manufactured by Merck Group
Sourced in United States
Phosphorylase inhibitors are a type of lab equipment used to inhibit the activity of the enzyme phosphorylase. Phosphorylase plays a key role in the breakdown of glycogen, a process that generates glucose. Phosphorylase inhibitors can be used to study the regulation of glycogen metabolism in various biological systems.
Automatically generated - may contain errors
Lab products found in correlation
Molecular imager chemidoctm xrs system, by Bio-Rad (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (1 mentions)
Mtorc1, by Cell Signaling Technology (1 mentions)
Hrp anti rabbit antibody, by Beyotime (1 mentions)
Anti bdnf, by Abcam (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Lysis buffer, by Beyotime (1 mentions)
Mini protean tgx precast gel, by Bio-Rad (1 mentions)
Chemiluminescence imaging system, by Bio-Rad (1 mentions)
Anti c myc, by Cell Signaling Technology (1 mentions)
Anti sox2, by Cell Signaling Technology (1 mentions)
Anti oct4a, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Anti klf4, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Triethylammonium bicarbonate, by Merck Group (1 mentions)
Pancreatin, by Promega (1 mentions)
Acetonitrile, by Thermo Fisher Scientific (1 mentions)
4 protocols using phosphorylase inhibitor
1
Western Blot Protein Analysis Procedure
2
Molecular Profiling of mPFC in Rats
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Animals were sacrificed and brains were rapidly removed and frozen in liquid nitrogen. mPFC was dissected out bilaterally using a brain mold on ice. Homogenates of the dissected tissue were lysed using lysis buffer (Beyotime Biotechnology, China) containing protease inhibitors (Sigma, USA) and phosphorylase inhibitors (Sigma, USA). Electrophoresis was performed on 4–15% Mini-PROTEAN TGX precast gels (Bio-Rad, USA). Primary antibodies used included anti-BDNF (1:500, Abcam, Cat GR3227037-2, USA), -M2-AChR (1:500, Abcam, Cat GR50911-14, USA), -mTORC1 (1:500, Cell Signaling Technology, Cat 2983S, USA), -phospho-mTORC (1:500, Cell Signaling Technology, Cat 5536S, USA) and -β-Actin (1:1,000, Santa Cruz, Cat SC47778, USA). Secondary antibodies used included HRP anti-rabbit antibody (1:5,000, Beyotime, China) and HRP anti-mouse antibody (1:5,000, Beyotime, China). Bands were detected using a chemiluminescence imaging system (Bio-Rad, USA). Image J (NIH, USA) software was used to analyze band densitometry.
3
Protein Expression Analysis in Intestinal Cells
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Intestinal tissue and cells were lysed in RIPA lysis buffer (Beyotime, P0013B) with protease inhibitor cocktail (Sigma-Aldrich, P8340), phosphorylase inhibitor (Millipore, 361515) and PMSF (R&D Systems, 4486/50). Then the 4× loading buffer (which is free of SDS for CD47) was added into the cell lysate. The mixture was heated on 60–65 °C for 5 minutes for CD47 while others were heated on 95–99 °C for 8 minutes. Protein levels were analyzed via Western blot using selective antibodies and normalized against the level of GAPDH. The following antibodies were used: anti-c-Myc (Cell Signaling, 2840 s), anti-KLF4 (Cell Signaling, 4083 s), anti-Oct-4A (Cell Signaling, 2840 s), anti-Sox2 (Cell Signaling, 23064 s), anti-CD47 (R&D, BAF1866), anti-GAPDH (Santa Cruz Biotechnology, sc-32233). Quantification of bands was analyzed by software Image J software.
4
Traditional Chinese Herbal Medicine Evaluation
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Medicinal herbs: The Chinese herbal medicine used in this study included Acanthopanax senticosus (Rupr. Maxim.) harms (purchased from Wuchang County, Heilongjiang Province, with a growth period of six years), Cynanchum otophylum (purchased from Haozhou City, Anhui Province), and Uncaria rhynchophylla (Miq.) Miq. ex Havil (purchased from Jianhe County, Guizhou Province).The TUNEL kit (Jiancheng), BCA protein concentration assay kit (Solarbio), RIPA lysis buffer (Solarbio), 5 × protein loading buffer (Biosntech), SDS‒PAGE gel preparation kit (Biosharp), Tris Base (Sigma), sodium dodecyl sulfate (Nachuan), special nonfat milk powder for sealing (Pulilai Gene), Tween 20 (BIOFROXX), western secondary antibody removal solution (Beyotime), hypersensitive ECL luminescent liquid (Meilunbio), LC3B (Sanying), P53 (Abcam), AMPK (Abcam), mTOR (Abcam), BAX (Boaosen), Caspase-3 (Boaosen), GAPDH (Boaosen), protease inhibitor (Calbiochem), pancreatin (Promega), acetonitrile (Fisher Chemical), trifluoroacetic acid (Sigma Aldrich), formic acid (FLUKA), iodoacetamide (Sigma), dithiothreitol (Sigma), urea (Sigma), triethylammonium bicarbonate (Sigma), purified water (Fisher Chemical), BCA Kit (Biyuntian), TMT labeling kit (Thermo), and phosphorylase inhibitor (Millipore) were the chemicals used in our study.
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