Using 4–12% gradient gels, immunoblotting was performed through MES SDS-PAGE, as previously described [21 (link), 26 (link), 29 (link)–31 (link)]. Protein was normalized using the Bradford method. Primary antibodies used in the study included: anti-TFAM, transcription factor A, mitochondrial, 1:500 (SCBT, Dallas, TX), anti-GAPDH 1:1000 (Abcam, Cambridge, MA). The secondary antibody used in the study was a goat anti-mouse IgG (H&L) horseradish peroxidase (HRP) conjugate 1:10,000 (Thermo Fisher). GAPDH expression was used to normalize protein content. Chemiluminescence was measured through Radiance Chemiluminescent Substrate (Azure Biosystems, Dublin, CA), per manufacturer’s instructions and imaged using the G:Box Bioimaging system (Syngene, Frederick, MD). Images were captured through GeneSnap/GeneTools software (Syngene). Densitometry was analyzed using ImageJ and Fiji Software (NIH, Bethesda, MD). Data is represented as optical density with arbitrary units.
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