LV RNA Extraction, cDNA Synthesis, and Protein Analysis

DNA-free total RNA was extracted from LV samples by a standard protocol with an EZ-RNA-total RNA Isolation Kit (Biological Industries, Israel). cDNA synthesis (500 ng) was carried out with the Verso RT-PCR Kit (ABgene, USA) according to the manufacturer’s instructions. qRT-PCR was performed on a Bio-Rad iQ-Cycler using a SYBR Green PCR kit (Invitrogen, Israel). The primer sequences are provided in S1 Data. Western blot was performed according to standard procedure [3 (link)] using the following antibodies: mouse monoclonal anti-Cytochrome C (Cyt C) (Cat # ab14734, Abcam, USA), mouse monoclonal anti-Voltage-dependent anion channel (VDAC)1/porin (Cat # ab110325, Abcam, USA), and mouse monoclonal anti- sirtulin (Sirt)3 (Cat # Sc-365175, Santa Cruz Biotechnology, USA), rabbit anti-GAPDH (Cat # 37168, Abcam, USA) followed by goat anti-mouse horseradish peroxidase (Cat # 10015289, Jackson ImmunoResearch Laboratories, Inc. USA) and goat anti-rabbit horseradish peroxidase (Cat # 111035003, Jackson ImmunoResearch Laboratories, Inc. USA)