Lc3 1

The total proteins of CC cells were extracted by lysis buffer. The extracts were centrifugated at 12,000 ×g for 30 min at 4°C. The protein concentration was then detected by BCA kit. The proteins were separated by sodium dodecyl-sulfate polyacrylamide gel electrophoresis, followed by transferring to polyvinylidene fluoride (PVDF) membranes. The membranes were incubated in 5% nonfat milk at 25°C for 2 h, and then incubated with primary antibodies (LC3I, LC3II, Beclin1, and p62 were purchased from Thermo Fisher Scientific, Waltham, MA, USA) at 4°C overnight. After washing with tris-buffered saline with Tween-20 (TBST), the membranes were incubated with secondary antibodies for 1 h. The bands were visualized using enhanced chemiluminescence kit (Santa Cruz Biotechnology, CA, USA) and recorded with Tanon 5200 (Tanon, Shanghai, China).