Western Blotting Protein Expression Analysis

Western Blotting analyses were conducted as previously published (Baltanas et al., 2013 (link); Liu et al., 2019 (link); Castro et al., 2020 (link); Khong et al., 2020 (link)). 20 µg of protein per sample was separated using 12% Mini-Protean TGX Precast Gel, and transferred into PVDF membranes. Non-specific protein binding was blocked using 5% fat-free milk and 0.1% Tween-20 in PBS 1x for 1 h. Membranes were then hybridized with the specific primary antibodies overnight at 4°C (PPX 45 kDa; eGFP 25 kDa; CV-ATP5A 54 kDa; CIII-UQCRC2 48 kDa; CII-SDHB 29 kDa; CIV-COXII 22 kDa; CI-NDUFB8 18 kDa; all of them at 1:1,000 dilution). The levels of protein expression were normalized using β-actin content (47 kDa; 1:1000). The signal was detected using Piece ECL Substrate kit following manufacturer’s instructions and the Gel Doc XR Image System from BioRad (Hercules, California, US). Finally, the intensity of the signal was quantified by densitometry analysis using ImageJ software from NIH (Bethesda, Maryland, US).