35S-Methionine pulse labeling

Cells were grown overnight at room temperature to mid-log phase in CSM-methionine media containing 2% dextrose. The culture was concentrated to an OD₆₀₀ of 0.7 and incubated in a shaking water bath at 25°C. The vehicle or drug was added to the appropriate culture flask. A 1.2 mL sample of culture was transferred to a 1.6 mL screw top tube to label proteins made during the next 15 min. The labeling reaction was initiated by adding 1 µL of EasyTag L-[³⁵S]-Methionine from PerkinElmer at a stock concentration of 1 µCi/µL. The sample was mixed by vortexing and then placed into the 25°C shaking water bath. Labeling reactions were allowed to progress for 15 min, during which time samples were mixed by inversion every ~5 min. Labeling reactions were performed during 15 min intervals starting every 20 min. Samples were centrifuged at 13,200 rpm for 30 s, the supernatant was removed, and 250 µL of acid-washed beads were added before freezing on liquid nitrogen. Samples were prepared for SDS-PAGE similarly to samples described for western blotting in 140 µL of 1× sample buffer. After running the gel to the dye front, polyacrylamide gels were stained in R-250 Coomassie stain and then dried on Whatman paper using a Bio-Rad Gel Dryer. The dried gel was exposed film.