2.4. Western blot analysis

SW 948 cells were seeded at a density of 2 × 10⁶ cells per well 6-well plates and incubated with various concentrations of dihydroartiminisin for 48 h. The cells were harvested and washed twice with phosphate-buffered saline. RIPA lysis buffer kit (Best Bio, shanghai, China) was used for the extraction of total proteins from the cells on ice for 45 min. The cell lysates were collected and then centrifuged at 4 °C for 20 min at 12,000×g to remove the debris. Bicinchoninic acid protein assay kit (Multi sciences, Hangzhou, China) was used for the detection of concentration of proteins. The proteins were separated by electrophoresis using 10% SDS-PAGE gels and transferred to a PVDF membrane (Merck Millipore, Darmstadt, Germany). The membrane blocking was performed using BSA for 1 h followed by incubation with primary polyclonal antibodies overnight 4 °C. The membranes were the washed again with PBS before incubation with goat-anti-rabbit secondary antibodies at room temperature for 1 h. The protein bands were detected by using enhanced chemiluminescence Western blotting detection kit (Santa Cruz Biotechnology, Inc.).