35S-methionine incorporation assay

To measure global protein synthesis rates, we quantified the incorporation of ³⁵S-labeled methionine and cysteine into newly translated proteins. Cells were exposed to 15 min methionine starvation followed by a 45 min methionine incubation to label newly synthesized proteins, using 1 µl (1.25 µCi/ml) of EasyTag™ L-[35 S]-Methionine (PerkerElmer®) per well at 37 C, 95% humidity and 5% CO₂ conditions. After labeling, cells were washed twice in cold PBS, harvested and centrifuged in cold PBS to remove supernatant. Next, cell pellets were lysed in cell lysis buffer (Cell Signaling). Lysate was blotted on labeled 24 mm glass microfiber filters (GF/C Whatman®) that were presoaked in 20% TCA. Filters were placed in a vacuum manifold and incubated in 10% ice-cold TCA for 15 min, followed by 10% TCA at 90–95 °C for 10 min. Filters were washed twice with cold 2% TCA and then twice with 95% ethanol to remove TCA. Next, filters were air dried for 1 h at room temperature and placed in liquid scintillation cocktail (Ultima Gold, PerkerElmer®). Radioactivity was quantified using a scintillation counter (Tri-Carb 2900TR).