Radioimmunoprecipitation assay rapi buffer

Total protein was purified using Radioimmunoprecipitation assay (RAPI) buffer (Thermo fisher scientific). Concentration of protein samples were determined by Pierce BCA protein assay kit (Thermo fisher scientific). Protein samples mixed with loading buffer and NuPAGE sample reducing reagent (Thermo fisher scientific) were heat-shocked at 85℃ for 2 minutes before loaded into SDS-PAGE gel (Thermo fisher scientific). Electrophoresis was performed on a Novex mini-cell device (Invitrogen, Carlsbad, CA, USA) for 90 munities. The gel was then attached to a PVDF membrane (Thermo fisher scientific) to transfer the protein. After blocked in 1% BSA buffer for 1 hour, the membrane was incubated in diluted antibodies solution overnight at 4 ℃. Anti-KPNB1, anti-Cyclin B1 primary antibodies were purchased from Novus biologicals (Littleton, CO, USA). Anti-Cyclin D1 primary antibody was purchased from Santa Cruz Biotechnology. Anti-CDK1, anti-RCC1, anti-pRCC1, anti-β-actin, anti-Ki67, and anti-caspase 3 primary antibodies and all secondary antibodies were purchased from Cell signaling technology (Danvers, MA, USA). Targeted protein was imaged using SuperSignal West substrate (Thermo fisher scientific).