Quantification of MCU Protein Levels

Cell proteins were extracted after 96 h of H9c2 transfection, as previously described [18 (link)]. Briefly, after being washed with cold PBS, cell cultures were scraped, harvested, and resuspended in RIPA buffer. The samples were vortexed and sonicated on rounds of 10 sec sonication/10 sec rest and centrifuged at 15000 ×g 10 min. The protein concentration of the lysate was determined by Lowry protein assay. Protein lysates (30 μg/lane) were resolved on SDS-PAGE gel 10%, transferred onto PVDF membrane at 150 mA, 50 min, and incubated with anti-MCU antibody ab121499 (Abcam, Cambridge, MA, USA) 1 : 500 and washed three times for 10 min with PBS-Tw 0.5% and subsequently probed with secondary antibody anti-rabbit IgG conjugated with HRP (Millipore, Billerica, MA, USA) 1 : 5000 for 2 h at room temperature (RT). After washing three times for 10 min, protein-antibody blots were developed with Clarity™ Western ECL (Bio-Rad, Hercules, CA) and quantified by using a BioSpectrum 415 Image Acquisition System (UVP®, Upland, CA, USA). Anti-β-actin antibody (ab8229) 1 : 500 or anti-GAPDH antibody (ab9484) 1 : 500 (both of them from Abcam, Cambridge, MA, USA) was used as a loading control. The level of MCU expression was the ratio of intensities of the MCU-signal/β-actin signal normalized versus siRNA-Neg signal.