Easytag express protein labeling mix

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.

Metabolic labelling assays were performed as previously described [5 (link)] with the following modifications. Cells were serum starved in DMEM without methionine and cysteine (ThermoFisher Scientific, Cat. 21013024) for 45 min, and pulse-labelled in 100 μCi/mL EasyTag™ Express Protein Labeling Mix, [35S] (Perkin Elmer, Cat. NEG772) for 15 min (WT CFTR) or 1 hour (ΔF508-CFTR). Cells were rinsed with cold PBS containing 1mM MgCl₂ and 0.1mM CaCl₂ and chased in DMEM supplemented with 10% FBS, 2 mM glutamine, 2 mM methionine and 2 mM cysteine. Cells were harvested at 0, 1, 2, or 3 h in 500 μL RIPA buffer. Soluble material was separated by centrifugation at 20,000 x g for 5 min at 4°C. Protein concentrations were normalized using the BCA Protein Assay Kit, and pulse-labelled CFTR was isolated by immunoprecipitation with a mixture of CFTR monoclonal antibodies (Millipore, MAB3480 (1.5 μg) and MAB3484 (1.5 μg) per sample) and 50 μL protein G agarose beads (Millipore, Cat: 16–266) for 2.5 hours at 4°C. Beads were washed 3 times in RIPA buffer and proteins were eluted in Laemmli loading buffer. CFTR bands were separated by 6% SDS-PAGE and radioactive bands were detected using a Typhoon scanner (GE Healthcare), then quantified using ImageJ 1.46r software.