Ab9484

After transfection for 48 h, proteins were extracted from cultured cells using RIPA buffer (Thermo Fisher Scientific) containing protease inhibitor. Then, the protein concentration was assayed by BCA protein assay kit (Thermo Fisher Scientific). The high-temperature denatured protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at 100 V and transferred onto the polyvinylidene fluoride (PVDF) membranes (Amersham, USA). The membranes were incubated with primary antibodies overnight at 4°C after being blocked for 1 h. Then, the membranes were incubated with horseradish peroxidase labeled secondary antibody goat anti-rabbit IgG H&L (ab97051, 1:2000, Abcam, Cambridge, UK) at room temperature for 1 h. Afterwards, the membranes were washed with TBST buffer for three times. The primary antibodies included rabbit polyclonal anti-E2F7 (ab56022, 1:1000, Abcam) and rabbit polyclonal anti-GAPDH (ab9484, 1:1000, Abcam). All proteins were visualized using an optical luminometer (GE, USA). The relative expression of proteins was analyzed by using the Image Pro Plus 6.0 (Media Cybernetics, USA). All experiments were conducted in triplicate.

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.