Anti phospho akt

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).

Cells were lysed using radioimmunoprecipitation assay buffer (RIPA buffer; 50 mM Tris−HCl, 150 mM sodium chloride (NaCl), 1% NP−40, 0.1% sodium dodecyl sulfate (SDS), a protease inhibitor cocktail, 50 mM sodium fluoride, and 0.2 M sodium orthovanadate). After centrifugation (12,000× g for 15 min), the protein concentration of the supernatant was measured. The proteins in the cell lysate were measured using the Braford assay. Next, 20 μg of protein from each sample was electrophoresed on 10% acrylamide SDS−polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Millipore, Burlington, MA, USA). Membranes were then blocked in 5% skimmed milk for 1 h at room temperature and incubated with primary and secondary antibodies. The primary antibodies were anti−UCP1 (Abcam, Waltham, MA, USA), anti−PGC1α (Boster Bio, Pleasanton, CA, USA), anti−adipose triglyceride lipase (ATGL; Boster Bio), anti−phospho hormone−sensitive lipase (HSL; Cell Signaling, MA, USA), anti−TFAM (Invitrogen, Carlsbad, CA, USA), anti−NRF1 (Invitrogen), anti−AMPK (Cell Signaling), anti−phospho−AMPK (Cell Signaling), anti−AKT (Cell Signaling), anti−phospho−AKT, and β−actin (Sigma−Aldrich); proteins were visualized using chemiluminescent HRP substrate (Advansta Inc., San Jose, CA, USA).