3.4. Western Blot and Protein Degradation Assay

Cells were cultured under the indicated conditions as described in “Cell culture”. Cells were seeded in 6-well plates (3516, Corning, New York, NY, USA). Compounds were dissolved in DMSO for storage (10 mM). Following compound treatment, cells were washed with PBS, scraped from the plate, and lysed using cell lysis buffer (Applygen, Beijing, China). After the protein in the cell lysate was quantified via the BCA quantification method, the samples were placed in a metal bath at 100 °C and heated for 5 min. Cell lysates were separated using a 10% SDS–PAGE gel and transferred to PVDF membranes (Merck Millipore, Burlington, MA, USA). The PVDF membrane was blocked with 5% nonfat dry milk in TBST for one hour. The PVDF membrane was transferred to the antibody diluent and incubated overnight at 4 °C. PVDF membranes were washed three times with TBST for 5 min each and then incubated with the appropriate HRP-conjugated secondary antibody for 1 h at room temperature. After washing three more times with TBST, the PVDF membrane was stained with a hypersensitive ECL chemiluminescence reagent (Biodragon, Beijing, China) and then imaged with Tanon 5200. Grayscale quantitative analysis was performed using ImageJ software. Statistical analysis was performed using GraphPad Prism 8. DC₅₀ and D_max values were fitted using a four-parameter [inhibitor] versus response and reported directly from the Prism output. Mean ± SD and unpaired t tests were performed in GraphPad Prism.