Metabolic protein labeling and determination of protein synthesis rates

The effect of INK128 on protein synthesis was assessed by metabolic [³⁵S]-Methionine incorporation, cells labeled with 25 µCi of [³⁵S]-methionine/cysteine per mL (EasyTag Express Protein Labeling Mix, Perkin Elmer) in Met/Cys-free DMEM supplemented with gentamicin at 0.04 mg/mL, 5% FBS, and bovine insulin at 0.01 mg/mL, and incubated at 37°C for 30 min. Lysates were prepared using NP-40 buffer and specific activity of [³⁵S]-methionine/cysteine incorporation into nascent protein was determined by trichloroacetic acid (TCA) precipitation onto GF/C filters and liquid scintillation counting. Studies were repeated three times and data presented as mean, normalized to the control, with SEM.