Pvdf membrane

Protein lysates were isolated in radioimmunoprecipitation assay (RIPA) buffer. The BCA kit (Pierce, Thermo Fisher Scientific) was used to measure protein concentration. After separation on sulphate–polyacrylamide gel electrophoresis, the protein samples were transferred to PVDF membranes (BD Biosciences, Clayton, VIC, Australia). Membranes were incubated with primary antibodies (1:1,000) at 4°C overnight. The following monoclonal antibodies were used: anti-Bcl-2 (sc-7382), anti-Bax (sc-20067), and anti-p-Akt (sc-293125) (Santa Cruz Biotechnology). Secondary antibodies conjugated with the horseradish peroxidase enzyme were employed. The protein bands were visualized using an enhanced chemiluminescence kit (ECL, Amersham, United Kingdom) and the band intensities were analyzed using ImageJ.

After 48 h of transfection with miR-629-mimic and miR-629-inhibitor (400 nM), Capan-2 cells were harvested, and then 80 pg of total protein extract was separated on 10% SDS-PAGE gels and transferred to PVDF membranes (BD Biosciences). The membrane was probed with monoclonal anti-FOXO3 and anti-p21 antibodies (1 : 500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), as well as with anti-Cyclin D1 (1 : 500, Cell Signaling, Shanghai, China), anti-AKT, anti-phospho-AKT, anti-GSK3β, anti-phospho-GSK3β, and anti-β-actin (1 : 1000, Sigma–Aldrich, St.Louis, MO) antibodies. The membrane was further probed with peroxidase-conjugated secondary antibodies at optimized concentrations, and the protein bands were visualized using enhanced chemiluminescence (Amersham Pharmacia Corp., Piscataway, NJ, USA).

The cells were lysed as described previously [22 (link)]. The protein concentration was measured using BCA protein assay kit (Pierce, Rockford, IL). Total proteins (60 μg) were separated in SDS/polyacrylamide gels (10% gels) (Sigma–Aldrich, St. Louis, MO) and then transferred on to PVDF membranes (BD Pharmingen, San Diego, CA). After blocking with 5% non-fat milk at room temperature for 1 h, the PVDF membranes were incubated primary antibodies against cleaved-Caspase-3, Bax, Bcl-2, PTEN, p-AKT, AKT, p-mTOR, and mTOR at 4°C overnight. These antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.). β-actin (Sigma, St. Louis, MO, U.S.A.) was used as an internal control. Horseradish peroxidase-conjugated (HRP) antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) were used as the secondary antibodies. Subsequently, the protein bands were scanned on the X-ray film using the ECL detection system (PerkinElmer Life and Analytical Sciences, Boston, MA). The alpha Imager software (Alpha Innotech Corporation, San Leandro, CA) was used to measure relative intensity of each band on Western blots. The measurements were performed independently for at least three times with similar results.

Western blotting was carried out as described previously.^{45 (link)} Total protein was extracted from the cultured cells using RIPA lysis buffer (Beyotime, Nanjing, China), which was supplemented with a protease inhibitor cocktail according to the manufacturer’s instructions. The protein mixtures were centrifuged to remove cellular debris. The extracted proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred to polyvinylidene fluoride (PVDF) membranes (Amersham, Little Chalfont, UK). The PVDF membranes were blocked for 2 h at room temperature using 5% non-fat milk (BD Biosciences, San Jose, CA). The membranes were incubated at 4 °C overnight with the following primary antibodies: anti-OCN (1:1 000; Boster Biological Technology, Wuhan, China), anti-BSP (1:1 000; Boster Biological Technology), anti-ALP (1:1000; Proteintech Group, Wuhan, China), anti-RUNX2 (1:1 000; Proteintech Group), and anti-GAPDH (1:1 000; Boster Biological Technology). Subsequently, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (ZSGB-Bio) for 1 h at room temperature. The immunoreactive bands were visualized using an ECL chemiluminescence reagent (Solarbio). The relative intensities were analyzed and compared to their respective controls using ImageJ software (NIH, USA).