β action

The cells were transfected with miR-196a, miR-205, or along with pcDNA or pcDNA-GAS5 and cultured for 48 h. Then, the cells were harvested and lysed in ice-cold radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, Jiangsu, China) for 40 min. Protein concentration was quantified using an Enhanced BCA Protein Assay Kit (Beyotime). Equal amount of protein samples were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5% non-fat milk for 1 h at room temperature and then probed with primary antibodies forkhead box protein O1 (FOXO1; 1:2000 dilution; Cell Signaling Technology, Beverly, MA, USA), phosphatase and tensin homolog (PTEN; 1:2000 dilution; Cell Signaling Technology), and β-action (1:5000 dilution; Sigma) for overnight at 4°C, followed by incubation with horseradish peroxidase-conjugated mouse and rabbit secondary antibody (1:2000 dilution; Abcam, Cambridge, MA, USA) for 2 h at room temperature. The signals were visualized using an enhanced chemiluminescence system (ECL ™ ; Amersham, Little Chalfont, UK) and analyzed using a FluorChem FC2 Imaging System (Alpha Innotech, San Leandro, CA, USA).