Foxo4

Western blots used the primary antibodies directed against the following proteins: Foxo1, Foxo3, Foxo4, α-tubulin from Cell Signaling (#2880, 2497, 9472, and 2144). We fixed and processed tissues for immunohistochemistry as described (Kitamura et al., 2009 (link)). We applied perfused fixation, and antigen retrieval for nuclear transcription factor detection (Nacalai USA) (Talchai et al., 2012b (link)). We used anti-Foxo1 (Cell Signaling #2880), anti-insulin and anti-glucagon from Dako (#A056401-2, A056501-2) for primary antibodies and FITC-, Cy3-, and Alexa-conjugated donkey secondary antibodies (Jackson Immunoresearch Laboratories, and Molecular Probes)(Kitamura et al., 2009 (link)). Antibodies to Pcsk1, Pcsk2, Glut-2 and Pdx-1 were from EMD Millipore (#AB10553, AB15610, 07-1402, 061384). For β-cell morphometry, 4 pancreatic sections from 4 mice from each genotype were sampled 150 μm apart, and used for a two-tailed paired Student’s t-test analysis. Total pancreas area was captured at 100x, and immunostaining area at 200x (Talchai et al., 2012b (link)). The ratio of immunoreactive area/pancreas area was converted from pixel to mm² and plotted as mm².

Total proteins were extracted using RIPA buffer (50 mM Tris-HCl, pH 8.0, 0.5% deoxycholic acid, 1% NP-40, 0.1% sodium dodecyl sulfate and 0.5 M NaCl) supplemented with protease inhibitor cocktails and phosphatase inhibitors (Pierce). Protein concentration was measured using BCA protein assay (Pierce). The lysates were separated by NuPAGE Bis-Tris gel (Invitrogen) and transferred to nitrocellulose membranes (Bio-Rad). Membranes were incubated in Blocking buffer (Li-Cor) for 30 min at room temperature; primary antibody (1:1000 dilution) at 4°C overnight; and then secondary antibody (1:10000 dilution) conjugated with IRDye 800CW or IRDye 680RD (Li-Cor) for 1 hour at room temperature. Immunoblots were scanned using an Odyssey Clx imaging system (Li-Cor Biosciences) and protein band intensity was measured using Image studio software (version 5.2). Each phosphokinase is paired with its steady-state control protein and loading is validated by actin levels. Antibodies were purchased from Cell Signaling (FOXO4, FKBP5, pAMPKα (T172), AMPK, pmTOR (S2448), mTOR, pP70S6K (T389), P70S6K, pS6 (S240/244), S6, pSTAT1 (Y701), STAT1, pSTAT3 (Y705), STAT3, pSTAT6 (Y641), STAT6, TBX21, GATA3), ThermoFisher Scientific (RORC), Abcam (FOXO4), and Millipore (Actin).
The unprocessed immunoblots are provided in Source Data.

Immediately following euthanasia, LD samples were stored at −70°F until processing. Free intracellular AA concentrations in muscle were determined by high-performance liquid chromatography (HPLC; PICO-TAG reverse-phase column; Waters, Mildford, MA,) using an analytical method based on deproteinization and derivatization of AA with phenylisothiocyanate, as previously described (18 (link)).
Amino acid transporter and intracellular markers of protein degradation and autophagy were measured by immunoblotting analysis, as previously described (8 (link), 31 (link)). The antibodies used in the immunoblotting process were PKB (total and Ser473, Cell Signaling Technology, Beverly, MA), AMPK-α (total and Thr172, Cell Signaling Technology, Beverly, MA), FOXO1 (total protein, Santa Cruz Technology, Santa Cruz, CA; Ser256, Cell Signaling Technology), FOXO4 (total protein and Ser262, Cell Signaling Technology, Beverly, MA), pro-caspase 3 (total protein, Cell Signaling Technology, Beverly, MA), MuRF1 (ECM Biosciences, Versailles, KY), atrogin-1 (ECM Biosciences, Versailles, KY), LAT1 (Cell Signaling Technology, Beverly, MA), SNAT2 (Aviva System Biology, San Diego, CA.), LC3 (Cell Signaling Technology, Beverly, MA), β-actin (Santa Cruz Biotechnology, Santa Cruz, CA), α-Actin (40 KDa; Dako-Cytomation, Glostrup, Denmark) and LAMP-2 (Cell signaling Technology, Danvers, MA)