Nupage bis tris mini protein gels

Total protein was extracted using RIPA buffer (Sigma-Aldrich) and 1% Protease Inhibitor Cocktail (Sigma-Aldrich). Following centrifugation, protein lysates were collected, and the concentration was measured using the Quick Start™ Bradford Protein Assay (Biorad). Protein samples were loaded on 4–12% NuPAGE™ Bis-Tris Mini Protein Gels (ThermoFisher), and electrophoresis was conducted the XCell SureLock Mini-Cell Electrophoresis System (ThermoFisher). Protein was subsequently transferred to an Immobilon-FL PVDF membrane (Merck) using the Mini Protean II Trans-Blot Cell (Bio-Rad). Following transfer, membranes were cut at 50 kDa and 90 kDa to generate three pieces for staining with different antibodies. Membranes were blocked with 5% bovine serum albumin (BSA) in tris-buffered saline (TBS) for one hour at room temperature. The membrane was incubated in primary antibodies solution overnight at 4 °C, followed by incubation with secondary antibodies for 1.5 h at room temperature. Membranes were washed three times in TBS buffer supplemented with 0.1% TWEEN-20 and scanned using the Li-Cor Odyssey 9120 Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA). Original fluorescence images of Western blots are shown in Supplementary Figure S4. Antibodies used are listed in Supplementary Table S2.

For SDS–polyacrylamide gel electrophoresis (SDS-PAGE)/western blotting experiments, 20 μg of protein was loaded for either whole cell lysate or mitochondrial samples, while 30 μg of protein was used for cytosolic and vacuolar fractions. Proteins were separated on 4–20% stain-free gels (Bio-Rad) or 12% NuPAGE Bis-Tris mini protein gels (Thermo Fisher Scientific) and blotted onto a polyvinylidene difluoride membranes. Membranes were blocked for 1 h in 5% (w/v) nonfat milk dissolved in Tris-buffered saline with 0.1% (w/v) Tween 20 (TBST-milk), followed by overnight incubation with a primary antibody in TBST-milk or TBST- 5% (w/v) BSA at 4 °C. Primary antibodies were used at the following dilutions: Cox2, 1:50,000 (Abcam 110271); Por1, 1:100,000 (Abcam 110326); Pgk1, 1:50,000 (Life Technologies 459250), Sod1, 1:5000, and Vma2, 1:10,000 (Sigma H9658). Secondary antibodies (GE Healthcare) were used at 1:5000 for 1 h at room temperature. Membranes were developed using Western Lightning Plus-ECL (PerkinElmer) or SuperSignal West Femto (Thermo Fisher Scientific).