Gel 2000 image analyzer

After incubation for 12h in a 6-well plate, cells were treated with LAS, or TAX for another 48 h. Then, these cells were collected with RIPA buffer containing protease and phosphatase inhibitors and let still at 4°C for 10 min to obtain lysates. Cell lysates were centrifuged at 12000 rpm for 15 min at 4°C and the protein concentration of lysates was estimated via the bicinchoninic acid (BCA) assay. The same amount of protein was separated on SDS- polyacrylamide gel electrophoresis and transferred to PVDF mem- branes. The membranes were blocked with 5% skimmed milk in TBST at 37°C for 5 min, and then incubated with primary antibodies overnight at 4°C followed by incubation with secondary antibodies for 2 h at room temperature. Primary antibodies were purchased from Cell Signaling Technologies and all these antibodies were diluted at a ratio of 1:1000 with TBST containing 5% BSA. Goat polyclonal anti- rabbit IgG conjugated to HRP and goat polyclonal anti-mouse IgG con- jugated to HRP (Cell Signaling Technology, MA, USA) were used as secondary antibodies and all these antibodies were diluted at a ratio of 1:5000 with TBST. Enhanced chemiluminescence reagents (Millipore, MA, USA) were used for detection and exposed by Gel (2000) image analyzer (Bio-Rad, CA, USA).

Western blotting analysis was performed as the previous study.^{19 (link)} Antibodies used were as following: rabbit polyclonal anti-PARP, rabbit polyclonal anti-caspase-3, rabbit polyclonal anti-cleaved caspase-3, rabbit polyclonal anti-caspase-9, rabbit polyclonal anti-cleaved caspase-9, rabbit polyclonal anti-cyclin B1, mouse polyclonal anti-cdc2 and rabbit polyclonal anti-p-cdc2 (Tyr15) were purchased from Cell Signaling Technology (Beverly, MA, USA). Mouse polyclonal anti-MPM2 and rabbit polyclonal anti-BICD2 were commercially available from Millipore Corporation (Bedford, MA, USA), and rabbit polyclonal anti-FOXM1 antibody was purchased from ABclonal Technology (Wuhan, China). Mouse polyclonal anti-β-actin was purchased from Sunshine Biotechnology Ltd. Goat polyclonal anti-rabbit IgG conjugated to HRP and goat polyclonal anti-mouse IgG conjugated to HRP (Cell Signaling Technology) were used as secondary antibodies, and enhanced chemiluminescence reagents (Millipore) was used for detection and exposed by Gel 2000 image analyzer (Bio-Rad, Richmond, CA, USA).