Quantification of De Novo Protein Synthesis

De novo protein synthesis assays were performed using the methionine analogue L-azidohomoalanine (AHA)^{60 (link)}. Briefly, cells were starved for 30 min in methionine-free RPMI media containing 10% dialysed FBS and 0.2 mM L-cysteine. Cells were given a pulse with AHA (100 µM) and incubated for 2 h at 37 °C in a humidified 5% CO₂ incubator. Cells were washed in PBS, harvested in lysis buffer (1% SDS in 50 mM Tris-HCl pH 8.0) and boiled at 95 °C or 50 °C for OXPHOS proteins. Biotin labelling was performed using the Click-iT Protein Reaction Buffer kit (Invitrogen, C10276) as per manufacturer’s protocol, followed by streptavidin pull-down (Dynabeads M-280 streptavidin) and western blot analysis.