Non-radioactive SUnSET assay

To measure in-vitro protein synthesis, Surface sensing of translation (SUnSET), a non-radioactive method to monitor protein synthesis was employed (Schmidt et al., 2009). Cardiomyocytes seeded on six-well plates were pulsed with puromycin (1 μM) for 30 min prior to harvesting. Cells were washed twice with ice-cold PBS and lysed. Bradford assay was performed for protein quantification and 80 µg of protein was boiled in Laemmli Sample Buffer (Bio-Rad) supplemented with 5% β-mercaptoethanol for 5 min at 96°C. SDS-PAGE was performed, and the proteins were then transferred onto a 0.45 µm PVDF membrane (Amersham Hybond P, GE) by western blotting for 16 hr at 25V at 4°C. After 1 hr of blocking with 5% non-fat milk at room temperature, the membrane was incubated with anti-puromycin antibody (DSHB, University of Iowa) overnight at 4°C. Membrane was washed thrice with 1X TBST and incubated with anti-mouse secondary antibody for 1 hr at RT. Blot was washed thrice with 1X TBST and chemiluminescent signals were captured using BioRad Clarity ECL western Blotting Substrate in a chemiluminescence imager (Chemidoc Touch, Biorad).