Anti akt

Western blots were performed using anti-INPP4B (Abcam, UK), anti-E-cadherin (Abcam, UK), anti-N-cadherin (Abcam, UK), anti-twist (OriGene, USA), anti-slug (OriGene, USA), anti-Cleaved caspase-3 (Cell Signaling Technology, USA), anti-Cleaved PARP (Cell Signaling Technology, USA), anti-Phospho-Akt (Invitrogen, USA), anti-Akt (Invitrogen, USA), anti-Phospho-PI3K (Sigma-Aldrich, USA), anti-PI3K (OriGene, USA), anti-p53 (Invitrogen, USA) and anti-GAPDH (Immunology Consultants Laboratory, USA) as previously described.27 (link)

Cells were harvested and then lysed in RIPA buffer with protease inhibitors (Protease Inhibitor Cocktail, Sigma). For the detection of nuclear proteins, the nuclei of ARPE-19 cells were separated by the Nuclei Isolation Kit (Sigma). After the determination of protein concentration, 50 μg protein extracts were resolved in 12% SDS-PAGE and transferred to a PVDF membrane. After blocking by 5% non-fat milk for 1 h at room temperature, membranes were blotted in primary antibodies overnight at 4 °C, followed by the incubation with secondary antibodies conjugated with horseradish peroxidase (1: 3000 dilution; Abcam, Cambridge, MA, USA) for 1 h at 37 °C. The primary antibodies employed for blotting were as follows: anti-bax (1: 1000 dilution; Invitrogen, Carlsbad, CA, USA), anti-bcl-2 (1: 500 dilution; Abcam), anti-p-Akt (1: 1000 dilution; Invitrogen), anti-Akt (1: 1000 dilution; Invitrogen), anti-nuclear factor erythroid 2-related factor (Nrf2; 1: 500 dilution; Abcam), anti-heme oxygenase-1 (HO-1; 1: 500 dilution; Abcam), and anti-β-actin (1: 1000 dilution; Invitrogen). The signals were visualized using an ECL kit (Thermo Fisher Scientific) and Image J software (National Institutes of Health, NIH, Bethesda, MD, USA), respectively.