GRS was a mixture of ginsenoside Rb1, ruscogenin and schisandrin (6:0.75:6). Schisandrin and ginsenoside Rb1 were obtained from Zelang Bio-Technology Co., Ltd (Nanjing, China). Ruscogenin was seperated in the laboratory and the purity was higher than 99%. Fetal bovine serum (FBS) was received from ScienCell (Carlsbad, CA, USA). Dulbecco’s modified Eagle medium (DMEM) was purchased from GIBCO/BRL (Life Technologies, Carlsbad, CA, USA). N-acetyl cysteine (NAC) was purchased from Sigma-Aldrich (St. Louis, MO, USA). 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) was purchased from AMRESCO (Cleveland, OH, USA). The kits for determination of lactate dehydrogenase (LDH), the malondialdehyde (MDA) and the fluorescent kit for 2′, 7′-dichlorofluorescein diacetate (DCFH-DA) were obtained from Beyotime Institute of Biotechnology (Shanghai, China). Protease inhibitor, RIPA lysis buffer, and enhanced chemiluminescene (ECL) reagent were obtained from Vazyme Biotech (Nanjing, China). Antibody against GAPDH was purchased from Bioworld Technology (St. Louis Park, MN, USA), antibody against JNK, p-JNK, p38, p-p38, ERK1/2, p-ERK1/2 and Jak2, p-Jak2, Stat3, p-Stat3 were obtained from Cell Signaling Technology (Boston, MA, USA).
Protease inhibitor
Manufactured by Vazyme
Sourced in China, United States
Protease inhibitor is a lab equipment product that functions to inhibit the activity of proteases, which are enzymes that break down proteins. It is used in various research and analytical applications to prevent protein degradation and maintain the integrity of protein samples.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Vazyme (4 mentions)
Ripa ice cold lysis buffer, by Vazyme (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by ScienCell (1 mentions)
N acetylcysteine nac, by Merck Group (1 mentions)
P erk1 2, by Cell Signaling Technology (1 mentions)
P jak2, by Cell Signaling Technology (1 mentions)
Antibody against gapdh, by Bioworld Technology (1 mentions)
P stat3, by Cell Signaling Technology (1 mentions)
Dulbecco s modified eagle medium dmem, by Thermo Fisher Scientific (1 mentions)
Stat3, by Cell Signaling Technology (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Avantor (1 mentions)
P jnk, by Cell Signaling Technology (1 mentions)
Erk1 2, by Cell Signaling Technology (1 mentions)
Blebbistatin, by Thermo Fisher Scientific (1 mentions)
Ginsenoside rg1, by Merck Group (1 mentions)
Claudin 5, by Abcam (1 mentions)
Glial fibrillary acidic protein (gfap), by Abcam (1 mentions)
Il 1β, by Thermo Fisher Scientific (1 mentions)
Tsg101, by Abcam (1 mentions)
6 protocols using protease inhibitor
1
Ginsenoside Rb1, Ruscogenin, and Schisandrin Evaluation
2
Western Blot Analysis of Primary Hippocampal Neurons
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Primary hippocampal neurons were lysed at indicated time points with 40-μl RIPA ice-cold lysis buffer (Rockford, IL, USA) supplemented with protease inhibitor (Vazyme Biotech, Nanjing, China) for 30 min and centrifuged at 9,000 g for 10 min at 4°C. The protein concentrations were analyzed by the BCA (Rockford, IL, USA). Equal amounts of proteins were separated and transferred to a PVDF membrane (Millipore, USA). The membranes were blocked with 3% BSA in TBS/T and stained with primary antibodies (1:1,000) and antibodies for β-actin (1:10,000) overnight at 4°C. Membranes were then probed with peroxidase conjugated secondary antibody at a 1:10,000 dilution. The antigen-antibody complexes were detected with an ECL reagent (Rockford, IL, USA). Primary antibodies used for western blotting were mouse antibodies. Secondary antibodies used were peroxidase conjugated goat antimouse antibodies.
3
Immunoprecipitation of NMIIA Protein
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The treated THP-1 cells were lysed in a RIPA lysis buffer containing protease inhibitor (Vazyme Biotech, Nanjing, China). Anti-NMIIA antibody (Abcam, USA) together with protein A/G agarose, was used to immunoprecipitate NMIIA and associated proteins. Proteins were resolved by SDS-PAGE and the resulting bands were detected by Western blot analyses or stained with silver staining and then comparatively analyzed to identify specific binding proteins by MALDI-TOF-MS.
4
Molecular Mechanisms of Ginsenoside Rg1 in Inflammation
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IL-1β and blebbistatin were procured from PeproTech Inc. (NJ, USA) and Selleckchem (Houston, TX, USA), respectively. Ginsenoside Rg1, dimethyl sulfoxide (DMSO), and anti-HSP90 antibody were acquired from Sigma–Aldrich (St. Louis, MO, USA). Primary antibodies against TIMP3, HSP90, p-P65, and P65 were obtained from Cell Signaling Technology (Beverly, MA, USA). ZO-1, occludin, claudin-5, TSG101, and GFAP were procured from Abcam (Cambridge, MA, USA). Antibody against NMIIA was purchased from Proteintech (Wuhan, China). Antibodies against MMP-3, MMP-9, CD6, CD9, VEGF, GAPDH and β-actin were acquired from EnoGene BIO Inc. (Nanjing, China). Secondary antibodies conjugated with horseradish peroxidase (HRP) were obtained from Bioworld Technology Inc. (St. Louis Park, MN, USA). MMP-1 antibody was acquired from Boster Biological Technology (Wuhan, China). Enhanced chemiluminescene (ECL) reagents, RIPA lysis buffer, and protease inhibitor were procured from Vazyme Biotech (Nanjing, China).
5
Evaluating Mitochondrial Function in Cardiac Cells
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QSYQ were provided by Tasly Pharmaceutical Co., Ltd. (Tianjin, China) with the production batch number of 200402. The biochemical kits for detection of lactate dehydrogenase (LDH), malondialdehyde (MDA), glutathione (GSH) and tissue iron determination were obtained from Nanjing Jian Cheng Biotechnology Company (Jiangsu, China). The ELISA kits for detection of precursor brain natriuretic peptide (NT-pro BNP) and 4-hydroxynonenal (4-HNE) were obtained from Shanghai Heng Yuan Biotechnology Company (Shanghai, China). RIPA lysis buffer, protease inhibitor, and enhanced chemiluminescence (ECL) reagent were purchased from Vazyme Biotech Co., Ltd. (Jiangsu, China). Hoechst33342 staining kit was provided by Beyotime (Nanjing, China). Antibody against β-actin were provided by Proteintech Group (IL, USA) and antibody against PGC-1α, TFAM, Nrf1 were purchased from Santa Cruz (CA, USA). Antibody against FHC, GPX4, DRP1, phospho-DRP1, MFN-2 and OPA1 were obtained from Proteintech Group (IL, USA) and antibody against ACSL4 were obtained from Abclonal Proteinch (Wuhan, China).
6
Quantitative Protein Expression Analysis
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Cells were lysed with 40 μL RIPA ice-cold lysis buffer (Rockford, IL, USA) supplemented with protease inhibitor (Vazyme Biotech, Nanjing, China) for 30 min at indicated time points. The protein concentrations in the whole cell lysates were determined using the BCA (Rockford, IL, USA) method after centrifugation at 7000 g for 10 min at 4°C. The protein samples were separated depending on their molecular weight on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) followed by a transfer to a polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA, USA). 5% nonfat powdered milk in Tris-buffered saline was used to block the membranes, which were stained with primary antibodies overnight at 4°C. After that, the membranes were probed with a 1:10,000 dilution of a peroxidase-labeled secondary antibody. An ECL reagent (Rockford, IL, USA) was used to detect the antigen-antibody complexes. Primary antibodies against β-actin were obtained from SIGMA St. (Louis, MO, USA), p21Waf1/Cip1, and p27 from Cell Signaling Technologies (Beverly, MA, USA). The ratio of drug-treated samples to control samples was used to calculate the relative expressions, which were normalized by the quantified level of β-actin expression.
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