Hrp conjugated goat anti rabbit and anti mouse igg antibodies

This study was approved by the Qilu Hospital Committee of Shan Dong University. Androgen-independent PCa cell lines (DU145 and PC-3) were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). All of the cells were grown in RPMI-1640 medium (Hyclone, Utah, USA) containing 10 % fetal calf serum (Gibco, CA, USA) at 37 °C in a 5 % CO₂ incubator. Baicalein was purchased from Selleck Chemicals (Houston, TX, USA) and was dissolved according to the manufacturer’ instructions. Antibodies against phospho-AKT (Ser473), AKT, phospho-mTOR, mTOR and GAPDH, and HRP-conjugated goat anti-rabbit and anti-mouse IgG antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA). Antibodies against cleaved PARP, Bax, Bcl-2, and survivin were obtained from Abcam (Abcam, Cambridge, UK).

Soluble whole-cell lysates of myoblasts were prepared using lysis buffer containing radioimmunoprecipitation assay (RIPA) buffer supplemented with 0.2 nmol/L PMSF (Complete, Roche, Shanghai, China). The lysates were denatured at 95 °C for 5 min and 10 mg of protein samples were subjected to 10% polyacrylamide gel. Blots were transferred to PVDF membranes and incubated in the primary antibodies, including DESMIN (1:50, DSHB, Iowa City, IA, USA), mouse, TET2 (1:400, Abcam, Fremont, CA, USA), MyHC (1:50, DSHB, Iowa City, IA, USA), α-TUBLIN (1:5000, Novus Biologicals Littleton, CO, USA). HRP-conjugated goat anti-rabbit and anti-mouse IgG antibodies (1:5000, Cell Signaling, Beverly, MA, USA) were used as secondary antibodies, respectively. Then, blots were detected using ECL reagents (Thermo Fisher Scientifc, Waltham, MA, USA). The quantity of detected proteins was normalized to that of α-TUBLIN using ImageJ 1.8 software (National Institutes of Health, Bethesda, MD, USA).