Cyclin b1

The total protein was extracted with a RIPA lysis buffer, and its concentration was determined with a BCA kit (Solarbio). The polyacrylamide gel was prepared in advance, and its concentration was determined by the size of the protein to be detected. The protein sample was loaded into a polyacrylamide gel, followed by electrophoresis for 2–3 h. Afterward, the protein was transferred onto a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA), and skim milk was used to block the heterogenetic antigens. Subsequently, the membrane loaded with protein was incubated with primary antibody at 4°C in the dark overnight. After rinsing with TBST buffer, the membrane was incubated with a secondary antibody labeled with HRP, and reacted with ECL reagent (Solarbio) for several minutes, followed by signal exposure in the dark. The antibody information was shown in the following: RASIP1 (1:1000; Affinity, Changzhou, Jiangsu, China), cyclin E1 (1:1000; Affinity), cyclin B1 (1:1000; Affinity), cyclin D1 (1:1000; Affinity), p27 (1:1000; Affinity), matrix metalloproteinase 2 (MMP2) (1:2000; Novus Biologicals, Littleton, CO, USA), MMP9 (1:1000; Affinity), cleaved caspase-3 (1:1000; Affinity), Bax (1:1000; Affinity), Bad (1:1000; Affinity), Bcl-xl (1:1000; Affinity), FOXO3 (1:500; Affinity), GAPDH (1:10000; Affinity).

Total protein lysates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a 0.22 μm polyvinylidene difluoride membrane (Millipore, Billerica, MA). They were then incubated with specific antibodies according to the manufacturer’s protocol. The GAPDH antibody was used as the control. The primary antibodies were as follows: FOXO3a (Abways, CY5079, Shanghai, P.R. China); p-FOXO3a (Abways, CY5562, Shanghai, P.R. China); PI3K (Abways, CY5355, Shanghai, P.R. China); p-PI3K (Abways, CY6427, Shanghai, P.R. China); GAPDH (Abways, AB0037, Shanghai, P.R. China); AKT (Affinity Biosciences, AF6261, USA); p-AKT (Affinity Biosciences, AF016, USA); NF-κB (Affinity Biosciences, AF5006, USA); p-NF-κB (Affinity Biosciences, AF2006, USA); BCL-6 (Affinity Biosciences, DF2903, USA); c-Myb (Affinity Biosciences, AF6136, USA); p53 (Affinity Biosciences, AF0879, USA); cylinG2 (Affinity Biosciences, DF2284, USA); cyclin B1 (Affinity Biosciences, AF6188, USA).