Crude mitochondria, cell pellets, eWAT and BAT were lysed in RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 12 mM deoxycholic acid, 0.5% Nonidet P-40 and protease and phosphatase inhibitors). Protein samples were used for SDS-PAGE followed by Western blotting. Nitrocellulose membranes were stained with primary antibodies against β-Actin, FoxO1 (Santa Cruz Biotechnologies), vDAC, Ucp1 (Abcam, Cambridge, UK), pHSL, PKA-Ser-Substrates (Cell Signaling Technologies, Danver, MA, USA), GSH (Enzo Lifescience, Farmingdale, NY, USA) and 4-hydroxy-2-nonenal (4-HNE) histidine (gently donated by Prof. Uchida, School of Bioagricultural Sciences, Nagoya University, Japan) all diluted 1:1000. Afterward, the membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody, and immunoreactive bands were detected by a Fluorchem Imaging System upon staining with ECL Selected Western Blotting Detection Reagent (GE Healthcare, Pittsburgh, PA, USA). Immunoblots reported in the figures are representative of at least three experiments that gave similar results.
Glutathione (gsh)
Manufactured by Enzo Life Sciences
Sourced in United States
The GSH product is a laboratory equipment item designed for scientific research and analysis. It serves the core function of measuring and quantifying the levels of glutathione (GSH) in various biological samples. The GSH product provides accurate and reliable data on this important antioxidant molecule, without making any claims about its intended use or applications.
Automatically generated - may contain errors
2 protocols using glutathione (gsh)
1
Mitochondrial Protein Analysis Protocol
2
Determining Cellular Glutathione Levels
Check if the same lab product or an alternative is used in the 5 most similar protocols
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The re-plated 3T3-L1 adipocytes were transfected with siFabp4 or siControl. At 48 h after transfection, the cells were washed with PBS and then scraped. A small part of the samples was utilized to determine protein content by the BCA method as described above. An equal volume of 5% meta-phosphoric acid (SIGMA) was added to each sample. After vortexing and sonication, the samples were centrifuged at 3000×g for 10 min at 4 °C. GSH content was determined using a BIOXYTECH GSH-400 kit (OXISResearch, Portland, CA, USA) according to the manufacture’s protocol. The standard curve was prepared using the purified GSH (Enzo Life Sciences, Farmingdale, NY, USA). The results were shown as an average GSH content per mg protein in three independent examinations.
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