Bca protein assay

After being exposed to curdepsidone A, HeLa cells were collected and lysed in RIPA buffer (Biotech Well, Shanghai, China) on ice. After quantified with the BCA protein assay (Elabscience, Wuhan, China), each sample was added to 5 × loading buffer (Beyotime, Shanghai, China) and then heated at 100 °C to denature the protein. The proteins were electrotransferred to PVDF membranes (Millipore, Billerica, MA, USA) after the lysate proteins were separated via SDS-PAGE. The membranes were hybridized with the suitable primary antibodies in the blocking solution for an entire night at 4 °C after blocking for 2 h. After that, the membranes underwent three TBST washes, one hour of incubation at 37 °C with a suitable secondary antibody, and another three TBST washes. Lastly, the ECL Chemiluminescence Kit (Beyotime, Shanghai, China) was used to detect protein signals on BG-gdsAUTO 720 (Baygene, Beijing, China). The expression of GADPH is regarded as a loading control. The primary antibodies against p-PI3K and p-mTOR were purchased from Cell Signaling Technology. Caspase3, cl-Caspase3, caspase7, cyto-c, Apaf-1, LC3, PI3K, and mTOR were purchased from Abcam. GAPDH, CDK4, Cyclin D1, PARP, Bcl-2, Bax, Caspase9, Beclin-1, p62, AKT, and p-AKT were purchased from Proteintech.