Phosphatases

Whole-cell proteins were extracted in lysis buffer (150 mM NaCl, 25 mM Tris pH7.5, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS) supplemented with inhibitors of proteases (Roche) and phosphatases (Sigma). Equal amounts of proteins were resolved on SDS-PAGE 4–12% (Bio-Rad) and transferred onto PVDF membranes. Membranes were blocked using 5% non-fat dry milk in TBS-Tween 0.1% (TBST) for 1 h at room and probed with primary antibodies overnight at 4 °C. The list of antibodies is available in Supplementary Table 1. After four washes in TBST, membranes were incubated for 1 h with HRP-conjugated secondary antibodies at 1:5,000 (Bio-Rad). After four more washes, immunoblots were developed by enhanced chemiluminescence. When required, PVDF membranes were stripped in 62.5 mM Tris HCl, pH6.8, 2% SDS, and 0.8% β-mercaptoethanol.

Tissue proteins were extracted using 300 µL of radioimmunoprecipitation assay (RIPA) buffer (25 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1.0% sodium deoxycholate, 0.1% SDS) in the presence of a cocktail of protease inhibitor (Roche Diagnostics, Basel, Switzerland)) and phosphatases (Roche Diagnostics, Basel, Switzerland)) and frozen at −80 °C. Protein concentration was determined using BCA Protein assay kit Protein (Sigma, St. Louis, MO, USA) with a standard curve of bovine serum albumin. A total of 30 µg of each sample was separated by electrophoresis in polyacrylamide gels (Tris-glycine SDS-polyacrylamide 10%) and electrotransferred to polyvinylidene fluoride (PVDF) membranes (0.45 µm). The primary antibodies and their dilution were FKBP5 1:1000 (12210S, Cell Signaling), G6Pase 1:500 (83690, Abcam, Cambridge, United Kingdom), and GAPDH 1:5000 (MAB374, Sigma). GAPDH was used as a loading control. The images were scanned using C-Digit and quantified using Image Studio Digits (LICOR Bioscience, Lincon, NE, USA).