Biomasher sp

One gram of cheese sample was homogenized in 2 mL ultrapure water with BioMasher SP (Nippi, Tokyo, Japan) and centrifuged for 5 min at 20,000 × g at 20°C. The solution was treated with a twofold volume of 5% trichloroacetic acid solution for deproteinization and centrifuged for 10 min at 20,000 × g at 20°C. The supernatant was filtered through an Ultrafree^®-MC Centrifugal Filter Unit (pore size, 0.2 μm; EDM Millipore, Billerica, MA, United States) by centrifuging for 5 min at 16,000 × g at 20°C prior to analysis. Organic acids and free amino acids were analyzed using HPLC, as previously described (Suzuki et al., 2021 (link)).

The frozen liver samples were thawed and a RIPA buffer containing Halt^TM Protease Inhibitor Cocktail was added to the samples at a volume of 4 mL per 1 g wet tissue weight. The tissue samples were homogenized using a Power Masher II and Bio Masher SP (Nippi, Tokyo, Japan). The homogenates were centrifuged at 13,000 g for 5 min at 4 °C and the supernatants were obtained for western blotting. The protein concentration of each sample was measured using a DC protein assay kit (Bio-Rad, Hercules, CA, USA). The samples of the supernatants mixed with EzApply (ATTO, Tokyo, Japan) were heated at 95 °C for 5 min and applied to 10% SDS polyacrylamide gels, and then the separated proteins were transferred to nitrocellulose membranes (Bio-Rad). After blocking for 30 min at room temperature with 3% skim milk in phosphate-buffered saline containing 0.05% Tween 20, the membranes were incubated overnight at 4 °C with rabbit antibodies against GPx-1, GPx-4 (1:1000, Abcam, Cambridge, UK) and β-actin (1:1000, Cell Signaling, Danvers, MA, USA) diluted by Can Get Signal I (TOYOBO Co. Ltd., Osaka, Japan). The membranes were subsequently incubated for 1 h at room temperature with peroxidase-conjugated anti-rabbit IgG antibody (1:5000, Cell Signaling) diluted by Can Get Signal II (TOYOBO Co. Ltd.). Immune complexes were visualized by an enhanced Immobilon™ Western (Merck Millipore, Bedford, MA, USA).

Whole-lung extracts were prepared from male mice lungs with nine volumes of ice-cold buffer (pH 7.4:5 mM phosphate buffer containing a complete protease inhibitor cocktail (Nacalai Tesque, Kyoto, Japan)) using Biomasher SP (Nippi Inc., Tokyo, Japan) for the thiobarbituric acid-reactive substances (TBA-RS) assay. Cytoplasmic and nuclear extracts were prepared using NE-PER™ nuclear and cytoplasmic extraction reagents (the protease and phosphatase inhibitors were added to CER I and NER) (Thermo Scientific, Rockford, IL, USA) using Biomasher II, according to the manufacturer’s instructions. Membrane extracts were prepared using the Mem-PER™ Plus Membrane Protein Extraction Kit (Thermo Scientific, Rockford, IL, USA) and Biomasher II according to the manufacturer’s instructions.