4.10. Pulse-Chase and Immunoprecipitation

Forty-eight hours post-transfection, cells were starved for 20 min in methionine/cysteine free media (Sigma-Aldrich, St. Louis, MO, USA), pulse labeled for 15 min with 50 mCi of [³⁵S]-methionine/cysteine (EasyTag™ EXPRESS³⁵S Protein Labeling Mix, PerkinElmer, Waltham, MA, USA) and chased for the indicated time points. Cell lysates (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% NP-40) and cell media were incubated with the appropriate antibodies ON at 4 °C for immunoprecipitation of the protein of interest. Immunoprecipitation proceeded as described above. Eluates were separated in reducing or non-reducing conditions and proteins were visualized by autoradiography. For experiments using 10 µM MG132 the inhibitor was added 1 h prior to starvation and maintained during starvation, pulse and chase time points. Band densitometry analysis was performed using ImageJ software (NIH, Bethesda, MD, USA).