Enhanced chemiluminescence kit

Total protein of the transfected OC cells was extracted using radio-immunoprecipitation assay (RIPA) lysis buffer (Beyotime, Shanghai, China), and the concentration of protein was evaluated by a bicinchoninic acid (BCA) protein assay kit (Beyotime, Shanghai, China). After the protein samples were mixed with loading buffer and denatured, the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransfered to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were subsequently blocked with 5% skimmed milk for 2 h at ambient temperature and incubated with specific primary antibodies overnight at 4 °C. Next day, the membranes were washed and incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG H&L (1:5000, ab205718, Abcam, Shanghai, China) for 1 h at ambient temperature. The protein bands were visualized by an enhanced chemiluminescence kit (Promega, Madison, WI, USA) and analyzed by Image-Pro Plus software. GAPDH was regarded as an internal reference. The primary antibodies were: anti-FBXW7 antibody (1:1000, ab109617, Abcam, Shanghai, China), anti-GAPDH antibody (1:2000, ab9485, Abcam).

Dulbecco’s modified Eagle’s medium (DMEM) was obtained from Hyclone (Logan, UT, USA); streptomycin, penicillin, PDGF, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), propidium iodide (PI) were obtained from Sigma-Aldrich (Louis, MO, USA); fetal bovine serum (FBS) was obtained from (Louis, MO, USA); The pcDNA3.1-PTEN (PTEN), control empty vector (control), miR-30b-5p inhibitors, miR-30b-5p mimics as well as their negative controls (NC) were bought from RiboBio (Guangzhou, China); Lipofectamine^TM 2000 was obtained from Invitrogen (Carlsbad, CA, USA). TRIzol reagent was obtained from Vazyme (Nanjing, China); PrimeScript RT Master Mix and SYBR PrimeScript™ miRNA RT-PCR kit were obtained from Takara (Dalian, China); the primers were provided by BGI (Shenzhen, China); 5-bromo-2-Deoxyuridine (BrdU) solution was obtained from BD Pharmingen (San Diego, CA, USA); RIPA lysis buffer was obtained from Beyotime (Shanghai, China); BCA protein detection kit was obtained from Pierce Chemicals Co. Ltd. (Rockford, IL, USA); the antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA); enhanced chemiluminescence kit was obtained from Promega (Madison, WI, USA).