S6 ps240 244

Epidermis was separated from the dermis and dissociated with a MixerMill homogenizer42 (link). For analysis of filaggrin, loricrin and keratin epidermis was lysed in 4% SDS lysis buffer. Alternatively cells or tissues were lysed in radio immunoprecipitation assay (RIPA) buffer, containing protease inhibitor (Sigma-Aldrich) and phosphatase inhibitor (Roche). Protein concentration was determined by Micro BCA Protein Assay Kit (Thermo Scientific) and 20 μg protein per sample was subjected to SDS–PAGE (Invitrogen). Subsequently protein was blotted to PVDF membranes. After blocking (5% non-fat milk in TBST buffer), membranes were incubated with primary antibodies. Primary antibodies and their dilutions included (Supplementary Table 3): mTOR, Raptor, Rictor, Akt, Akt-pS473, Akt-pT308, GSK3α/β, GSK3α/β-pS9/21, FoxO1, FoxO1-pT24/32, FoxO1-pS265, S6K1-pT389, S6, S6-pS240/244, 4E-BP1-pT37/46 and PKCα (all from Cell Signaling); p63, PKCα-pS657 (Santa Cruz Biotech); loricrin, K10, filaggrin (all from Covance); K14 (PROGEN Biotechnik); β-actin, α-Tubulin (Sigma). After incubation with horseradish peroxidase (HRP)-conjugated anti-mouse, anti-rabbit, anti-guinea pig or anti-goat secondary antibodies (DAKO), the blot was developed with ECL substrate (Pierce) and X-ray film (Amersham Biosciences). Not cropped western blotting results are presented in Supplementary Figs 7 and 8.

Tissues or cells were homogenized in a lysis buffer containing 100 mM Tris (Merck, Burlington, Massachusetts) pH7.5, 2 mM EDTA (Sigma-Aldrich, St. Louis, Missouri), 2 mM EGTA (Sigma-Aldrich, St. Louis, Missouri), 150 mM NaCl (Sigma-Aldrich, St. Louis, Missouri), 1% Triton X-100 (Fluka Chemie, Buchs, Switzerland), cOmplete inhibitor cocktail (Sigma-Aldrich, St. Louis, Missouri) and PhosSTOP (Sigma-Aldrich, St. Louis, Missouri). Protein concentration was determined by the Bradford assay (Bio-rad), and equal amounts of protein were separated by SDS-PAGE, and transferred onto nitrocellulose membranes (GE Healthcare, Chicago, Illinois). Antibodies used in this study were as follows: AKT (Cat#4685 or Cat#2920), AKT-pS473 (Cat#4060), AKT-pT308 (Cat#13038), PRAS40-pT246 (Cat#2997), PRAS40 (Cat#2691), HK2 (Cat#2867), S6-pS240/244 (Cat#5364), S6 (Cat#2217), FASN (Cat#3189), ACC (Cat#3662), HA tag (Cat#3724) and ChREBP (Cat#58069) from Cell Signaling Technology (Danvers, Massachusetts), GLUT4 (Cat# NBP2-22214, Bio-Techne, Abingdon, United Kingdom), HSP90, (Cat#sc-13119, Santa Cruz Biotechnology, Dallas, Texas), CALNEXIN (Cat#ADI-SPA-860-F, Enzo Life Sciences, Farmingdale, New York) and ACTIN (Cat#MAB1501, Merck, Burlington, Massachusetts). For quantification, the specific signals were normalized to a loading control.