Anti kif26b

Total protein was isolated from the cultured cell lines using radioimmunoprecipitation assay lysis buffer (Beyotime Biotechnology, Shanghai, China). The protein concentration in the cell extracts was measured using a bicinchoninic acid protein assay kit (Beyotime Biotechnology) with bovine serum albumin as a standard. Equal amounts of protein (40 μg) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred onto polyvinylidene difluoride (PVDF) membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked in 5% fat-free milk and then incubated with the following primary antibodies at 4°C overnight: anti-KIF26B (Abcam, Cambridge, UK), anti-GAPDH (Abways, Shanghai, China), anti-Bcl-2 (Cell Signaling Technology, Danvers, MA, USA), anti-Bax (Cell Signaling Technology), anti-cyclin D1 (Cell Signaling Technology), anti-p-Rb (ser780) (Cell Signaling Technology), anti-cleaved caspase-3 (Cell Signaling Technology), anti-E-cadherin (Cell Signaling Technology), anti-N-cadherin (Cell Signaling Technology), anti-Vimentin (Cell Signaling Technology), and anti-Snail (Cell Signaling Technology); then, the membranes were incubated with the secondary detection antibody (Cell Signaling Technology) for 1 h at room temperature. Protein bands were visualized using an enhanced chemilumescent (ECL) reagent (Pierce Biotechnology, Waltham, MA, USA).