Western Blot analyses

For protein isolation H9c2 and HL-1 cells were grown in 6 well plates and treated as desired. Cells were washed in PBS and lysed in 50 μl cell lysis buffer (#9803, Cell Signaling Technology) supplemented with protease (Complete Protease Inhibitor Cocktail Tablets, Roche Diagnostics) and phosphatase (PhosSTOP Phosphatase Inhibitor Cocktail Tablets, Roche) inhibitors for 10 min on ice. Cells were mechanically disrupted using cell scrapers, the lysate was transferred to a reaction tube and centrifuged at 12,000 × g for 10 min at 4°C. Protein concentration in the supernatant was determined by a modified Lowry assay (DC Protein Assay, BIO-RAD). For samples on the same gel equal protein amounts (usually 20 μg) were loaded, separated using denaturing polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto nitrocellulose (GE Healthcare) or PVDF (Millipore) membranes. Membranes were blocked for 1 h in 5% non-fat dry milk (Carl Roth) in TBS-T and incubated with primary antibodies at 4°C over night. A list of primary antibodies used in this study is provided in Supplementary Table S1. Antibodies against GAPDH, α-Tubulin and Vinculin were used for loading control and for normalization upon densitometric quantifications. Secondary detection was performed using horseradish peroxidase (HRP)-linked secondary antibodies (Cell Signaling Technology, #7074 and #7076, 1:2,000). Standard enhanced chemiluminescence (ECL) reaction was used for abundant proteins whereas weakly expressed proteins were detected using the SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific). ECL signals were imaged with the ChemiDoc XRS+ (BIO-RAD) system. Intensity of detected protein bands was quantified by densitometry using ImageJ (https://imagej.nih.gov/ij/) or Image Lab (BIO-RAD) software. Western blot membranes were usually cut horizontally based on the visible bands of the molecular size marker to allow simultaneous detection of multiple proteins of different molecular weight. As a result, the same loading control serves for normalization of various proteins that were run on the same gel and detected on the same membrane. Therefore, identical blot images for loading controls might appear in different figure panels showing western blot data.