Fluorescent conjugated anti mouse or anti rabbit secondary antibodies

Total proteins from mouse soleus tissue or cultured cells were extracted in Cell lysis buffer (Cell Signaling) completed with protease inhibitors (25 mg/ml aprotinin; 10 mg/ml leupeptin; 1 mM 4-[2-aminoethyl]-benzene sulfonylfluoride hydrochloride; 2 mM Na3VO4). The lysates were sonicated (3 pulses of 10 s at 30% amplitude) and protein concentration was measured with the Bicinchoninic Acid Assay protein assay (Pierce). Protein extracts were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes (Invitrogen). Blocking and antibody incubations were performed in 5% bovine serum albumin. Membranes were incubated with fluorescent-conjugated anti-mouse or anti-rabbit secondary antibodies (BioRad) 1h at RT. Antibody detection was imaged using the ChemiDoc Touch Imaging System (BioRad). Quantification was done using ImageJ software.

Total proteins were isolated from cultured cells, cardiac sections, or isolated cardiomyocytes with extraction buffer (Cell Signaling) containing protease inhibitors (25 mg/ml aprotinin, 10 mg/ml leupeptin, 1 mM 4-[2-aminoethyl]-benzene sulfonyl fluoride hydrochloride and 2 mM Na₃VO₄). The lysates were sonicated (3 pulses of 10 s at 30% amplitude). Cytosolic and nuclear fractions were prepared using NE-PER Nuclear and Cytosolic Extraction Reagents (Thermo Fisher Scientific) according to the manufacturer’s instructions. Sample protein content was determined by the bicinchoninic acid protein assay (Thermo Fisher Scientific). Protein extracts (10–20 µg) were analyzed by SDS–PAGE on a 10% gel and transferred onto nitrocellulose membranes (Invitrogen). After washing with Tris-buffered saline containing 1% Tween 20 (TBS-T), the membranes were blocked with 5% bovine serum albumin (BSA) in TBS-T for 1 h at room temperature and then incubated with the appropriate antibody overnight at 4 °C. Membranes were incubated with fluorescent-conjugated anti-mouse or anti-rabbit secondary antibodies (BioRad) 1 h at RT. Antibody detection was imaged using the ChemiDoc Imaging System and ImageLab software (BioRad). Quantification was performed using FIJI/ImageJ software.