Protein degradation analysis by western blot

Adherent cells were plated (100,000 cells/well in a 24-well plate) one day before the experiment. Cells were incubated with 250 μl of complete growth media with 10 nM LYTAC or controls for indicated time. Cells were then washed with DPBS 3 times and lysed with RIPA buffer supplemented with protease inhibitor cocktail (Roche), 0.1% Benzonase (Millipore-Sigma), and phosphatase inhibitor cocktail (Cell Signaling Technologies) on ice for 30 minutes. The cells were scraped, transferred to Eppendorf tubes, and spun down at 21,000g for 15 minutes at 4 °C. The supernatant was collected and the protein concentration was determined by BCA assay (Pierce). Equal amounts of lysates were loaded onto 4–12% Bis-Tris gel and separated by sodium dodecyl sulfate-polacrylamide 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 Odyssey Blocking Buffer (TBS) (LI-COR) for 1 hour at rt. The membrane was incubated with primary antibody overnight at 4 °C, washed 3 times with TBS-T. Subsequently, the membrane was incubated with secondary antibody for 1 hour at rt, and washed 3 times with TBS-T for visualization with an Odyssey CLx Imager (LI-COR). Image Studio (LI-COR) was used to quantify band intensities.