Metabolic Labeling and Protein Analysis

Cells were transfected with siRNAs in 6-well plates 48 h before metabolic labeling. Cells were then incubated for 5 min at 37 °C with 55 µCi/well of 35S-L-methionine and ³⁵S-L-cysteine Promix (Perkin Elmer). To validate the labeling efficiency, cycloheximide (100 mg/mL final) was added for 10 min prior to labeling. Cells were then washed with 1 ml of ice-cold PBS and lysed in 500 µL of RIPA buffer mixed with 1× final LDS Novex™ 4× Bolt™ loading buffer (ThermoFisher). Extracts were then run on precast Bis-Tris Bolt™ 4 to 12% acrylamide gels (ThermoFisher) before staining with the Simply Blue Safestain (Thermo) according to manufacturer’s guidelines to visualize total protein loading across lanes. The gel was then incubated in 30% ethanol, 10% acetic acid, and 5% glycerol for 1 h and dried at 75 °C for 90 min. ³⁵S radioactivity levels were measured using the Typhoon Phosphor imager.
For rescue experiments, cells were transfected with siRNAs in 96-well plates 48 h before HomoPropargylGlycine (HPG) metabolic labeling and treated or not with 100 ng/ml of doxycycline. Cells were then incubated for 30 min at 37 °C with 50 μM HPG in methionine/cysteine-free media. To validate the labeling efficiency, emetine (25 μg/mL final) was added during HPG labeling. Cells were then washed twice with PBS and HPG/Alexa Fluor 594 Click-it reaction was performed according to the manufacturer’s protocol (Life Technologies). Alexa Fluor 594 mean fluorescence intensities per cell were determined by HCS microscopy analyses as described above.