Protein degradation analysis by immunoblot

Cells were plated (75,000 cells per well in a 24-well plate) 1 day before the experiment. Cells were incubated with 250 μl of complete growth media with 10 nM LYTAC or controls for the indicated time. Cells were then washed with PBS three times and lysed with RIPA buffer supplemented with protease inhibitor cocktail (Roche), 0.1% Benzonase (Millipore-Sigma), and PhosStop (Roche) on ice for 30 min. The cell suspension was scraped and transferred to Eppendorf tubes, and spun down at 21,000 g for 15 min at 4°C. The supernatant was collected and the protein concentration was determined by BCA assay (Pierce). Equal amounts of lysates were loaded onto 4 to 12% Bis-Tris gel and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Then, the gel was transferred onto a nitrocellulose membrane and stained with REVERT Total Protein Stain (LI-COR), then blocked with Intercept Blocking Buffer (TBS) (LI-COR) for 1 hour at RT. The membrane was incubated with primary antibody overnight at 4°C, washed three times with TBS-T. Subsequently, the membrane was incubated with secondary antibody for 1 hour at RT and washed three times with TBS-T for visualization with an Odyssey CLx Imager (LI-COR). Image Studio (LI-COR) was used to quantify band intensities.