Western Blot Analysis of PTEN and p16

Western blot analysis was carried out as described before [5 (link)]. Briefly, the OSA cells and fibroblasts were lysed with CelLytic M lysis buffer (C2978, Sigma-Aldrich, St. Louis, MO, USA) in the presence of protease inhibitor (P8340, Sigma-Aldrich) and phosphatase cocktail inhibitor B (sc-45045, Santa Cruz Biotechnology, Dallas, TX, USA). Proteins were resolved on Bolt Bis-Tris 4–12% polyacrylamide gels (ThermoFisher Scientific, Waltham, MA, USA) and transferred to polyvinylidene difluoride membranes, incubated with 5% bovine serum albumin (BSA) for 2 h at room temperature, before being incubated with the primary antibodies at 4 °C overnight. The primary antibodies used were PTEN (Cell Signaling Technology, D 4.3, 1:2000), p16^INK4A (Santa Cruz Biotechnology, F-8, 1:500) and β-actin (Cell Signaling Technology, 8H10D10, 1:2000. The secondary antibodies were obtained from (LI-COR Biosciences, donkey anti-mouse; goat anti-rabbit (LI-COR Biosciences), both used at a 1:15,000 dilution and incubated with the blot for 1 h at room temperature. The membranes were visualized using the Odyssey^® M Imaging System (LI-COR Biosciences, Lincoln, NE, USA) and analyzed using Image Studio™ Lite software 5.2.5 (LI-COR Biosciences, Lincoln, NE, USA).