Western Blot Analysis of Phospho-AMPKα

Samples from the retina or the cell line were isolated and placed in lysis buffer, including protease inhibitor cocktail (Complete, EDTA-free; Roche, Mannheim, Germany) and phosphatase inhibitor cocktails 2 and 3 (Sigma-Aldrich) to prepare the lysate, and immunoblot analyses were performed as described elsewhere [2 (link)]. Briefly, the proteins were separated by electrophoresis and electrically transferred to a polyvinylidene fluoride membrane (Immobilon-P; Millipore; Bedford, MA, USA). The membrane was blocked with 5 % skim milk in Tris-buffered saline with Tween or TNB blocking buffer [0.1 M Tris–HCl, pH 7.5, 0.15 M NaCl, 0.5 % TSA Blocking Reagent (PerkinElmer Life Sciences; Waltham, MA, USA)], and then incubated overnight at 4 °C with a rabbit anti-phospho-AMPKα (1:1000; Cell Signaling Technology) or rabbit anti-AMPKα (1:200; Cell Signaling Technology) for normalization. Six samples for each group were analyzed.