Phosphatase inhibitor cocktail b

Nuclear extracts were prepared from 50 mg of soleus muscle using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific, Waltham, MA, USA). Complete Protease Inhibitor Cocktail (Santa-Cruz), Phosphatase Inhibitor Cocktail B (Santa Cruz), PMSF (1 mM), aprotinin (10 μg/mL), leupeptin (10 μg/mL), and pepstatin A (10 μg/mL) were used to maintain extract integrity and function. Nuclear extracts were dialyzed by means of Amicon Ultra-0.5 centrifuge filters (Millipore, Burlington, MA, USA).
The protein content of all samples was quantified twice using a Quick Start Bradford Protein Assay (Bio-Rad Laboratories) in order to calculate the optimal sample value for electrophoretic gel. The supernatant fluid was diluted with 2× sample buffer (5.4 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 10% β-mercaptoethanol, 0.02% bromphenol blue) and stored at −85 °C for immunoblot procedures.

The Abrams OSA cells (500,000/well in 6-well plate) were treated with either vehicle (0.1% DMSO) or sorafenib for 24 h. Cells were lysed with 250 μL of CelLytic M lysis buffer (C2978, Sigma-Aldrich, St. Louis, MO, USA), 2 μL of protease inhibitor (P8340, Sigma-Aldrich), and 2 μL of phosphatase cocktail inhibitor B (sc-45045, Santa Cruz, Dallas, TX, USA) according to manufacturer protocol. Protein concentrations were quantified with the QubitTM Protein Assay Kit. A total of 60 μg of protein per well was loaded on Bolt Bis-Tris 4–12% polyacrylamide gels (Thermo Fisher Scientific Inc.) and transferred to polyvinylidene difluoride membranes. The membranes were incubated with 5% bovine serum albumin (BSA) for 2 h at room temperature, then incubated with the following primary antibodies at 4 °C overnight to detect antigen: ERK (1:500), p-ERK (1:250), STAT3 (1:500), p-STAT3 (1:500), β-tubulin (1:4000) (Cell Signaling Technology). After three washes in tris-buffered saline with 0.05% Tween 20, the membranes were incubated with appropriate secondary antibody (donkey anti-mouse (1:15,000) or goat anti-rabbit (1:15,000)) for 1 h at room temperature. The membranes were visualized using the Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA) and analyzed using Image Studio™ Lite software (LI-COR, Lincoln, NE, USA).

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).