Western Blot Analysis of Autophagy Markers

WB was conducted as previously reported^{34 (link)}. In brief, cells were lysed in radioimmunoprecipitation assay buffer (Beyotime, China) containing 1% phenylmethylsulfonyl fluoride. Extracted protein concentration was measured by the bicinchoninic acid method and stored at −80 °C. A 25-µg protein sample was run on an 8–10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to a polyvinylidene difluoride membrane (Bio-Rad, USA). Afterward, the membrane was blocked in 5% nonfat milk (BD, USA) and incubated with the primary antibodies at 4 °C overnight. Finally, the membrane was incubated with the secondary antibodies and developed under the gel electrophoresis imager (Bio-Rad, USA). The primary antibodies of LC3 (ab192890), P62 (ab207305), and ATG5 (ab109490) were purchased from Abcam, while p53 (10442-1-AP), Zeb1 (21544-1-AP), and GAPDH (10494-1-AP) antibodies were purchased from Proteintech. The secondary antibody anti-Rabbit (SA00001-2) was also purchased from Proteintech.