Protein Synthesis Assay (SUnSET)

Protein synthesis was measured using the SUrface SEnsing of Translation (SUnSET) method which measures the incorporation of puromycin into nascent peptide chains (Schmidt et al., 2009; Goodman et al., 2011). Puromycin dihydrochloride (Sigma-Aldrich) was added to the cell treatment media (1 μM final concentration) for 30 min prior to cell lysis with a standard RIPA buffer. Twenty micrograms of protein were separated in a 10% polyacylamide gel until the dye-front was ∼2 cm from the bottom of the gel. Proteins were transferred to nitrocellulose membranes (BioRad) and subsequently incubated in TBST buffer containing 5% skim milk powder and incubated overnight with a monoclonal puromycin antibody (clone 12D10, EMD Millipore, Temecula, CA, United States, diluted 1:5000 in TBST). Membranes were then incubated for 1 h in TBST containing 5% skim milk plus a secondary mouse antibody (Sigma-Aldrich, 1:10000). The total lane density was analysed using Image J (NIH).