Western Blot Analysis of Mitochondrial Proteins

Western blot was performed according to the previously described methods [21 (link)] with minor modifications. Samples were extracted following homogenization in RIPA buffer, and protein concentrations were determined using a protein assay kit (Bio-Rad, Hercules, California, USA) with BSA as the standard. A total of 10 μg of protein was subjected to electrophoresis using 10%–12% SDS-polyacrylamide gel. Then, these proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane (Bio-Rad). The antigens were detected using primary antibodies (anti-POLG, diluted 1:1000, ab128899, Abcam; anti-SSBP1, diluted 1 μg/mL, AF6588, Bio-techne; anti-β-ACTIN, diluted 1:1000, ADI-CSA-335, Enzo; and anti-GAPDH, diluted 1:2500, ADI-CSA-335-E, Enzo), followed by secondary immunoglobulins conjugated to HRP (1:1000, Bio-Teche for SSBP1 and 1:10,000, GeneTex for POLG, β-ACTIN, and GAPDH), and an ECL reagent kit was used for visualization (pico EPD, Elpisbio, Daejeon, South Korea). The assay was triplicated, and the expression of the POLG protein was normalized with the β-ACTIN and GAPDH expression. The band intensities were completed using ImageJ software (National Institutes of Health, Bethesda, MA, USA).