Western Blot Analysis of FOXO3, LC3, and S6

FOXO3 western blots were performed as described previously [44 (link)]. Cells were lysed in Triton lysis buffer (50mM Tris-HCL pH 7.5, 100mM NaCl, 0.5 mM EDTA, 0.4% Triton, 50 mM NaF, 40 mM b-glycerophosphate, 1mM sodium orthovanadate, 1mM PMSF, 0.055 units/ml aprotinin). For LC3 western blots, cells were lysed in 150 mM NaCl, 1.0% Triton, 0.5% Sodium Deoxycholate, 0.05% SDS, 50mM Tris-HCL pH 8 and a cOmplete Mini Protease Inhibitor Cocktail Tablet (Sigma) by shaking at 4°C for 30 minutes. Lysates were centrifuged at max speed for 7 minutes (15 minutes for LC3) and supernatants transferred to new tubes. Protein concentration was quantified on a Qbit using the Qbit Protein Assay Kit (Life Technologies), followed by the addition of Laemmli sample buffer (2% SDS, 10% glycerol, 5% β-meracaptoethanol, 63 mM Tris-HCl pH 6.8, bromophenol blue). All antibodies were diluted in 5% powdered milk or BSA and primary antibody incubations were performed overnight at 4°C. Primary antibodies and concentrations used: Rabbit anti-LC3B 1:1000 (Novus Biologicals NB600-1384), Rabbit anti-FOXO3 1:1000 (CST 75D8), Rabbit anti-FOXO1 1:1000 (Abcam ab12161), Rabbit anti-pS6 1:1000 (CST 4858S), Mouse anti-S6 1:1000 (CST 2317S), Rabbit anti-Pink1 1:1000 (Novus BC100-494) Mouse anti-Actin 1:5000 (Novus Biologicals (NB100-74340).