Na pyrophosphate

Retinas and optic nerves were collected and separately lysed in 93 μl of Lysis Buffer solution (LB) composed by 1% Triton X-100 (Sigma-Aldrich, Milan, Italy, nr 9002-93-1), a complete set of protease inhibitors (Complete, Roche Diagnostics, Basel, Switzerland) and phosphatase inhibitors (Sigma, St. Louis, MO), N-ethylmaleimide (Sigma-Aldrich, Milan, Italy, nr 128-53-0), a buffer containing the following components (mM): TRIS acetate, 20; sucrose, 0.27; EDTA, 1; EGTA, 1; Na Orthovanadate, 1; NaF, 50; Na Pyrophosphate, 5; Na β- glycerophosphate, 10; and DTT, 1 (Sigma-Aldrich, Milan, Italy). Then samples were kept for 30 min on ice to allow protein extraction. Later a centrifugation step of 10 min at 10,000 r.p.m. was applied to the samples and the supernatant was collected and stored at −20 °C until needed.

After completion of the behavioral testing, the mice were anesthetized with isoflurane, decapitated and the brain removed. The hippocampus was isolated from the remainder of the brain and snap frozen in liquid nitrogen for later processing and protein assays. Protein samples were prepared from the whole hippocampus using standard protocols, as previously described [30 (link)]. Briefly, proteins were extracted by sonication in cold lysis buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.5% NP-40, a Complete Protease Inhibitor Cocktail Tablet (Roche Diagnostics, Mannheim, Germany), and a cocktail of phosphatase inhibitors (Na⁺ pyrophosphate, β-glycerophosphate, NaF, Na⁺ orthovanadate; all from Sigma-Aldrich). After 30-min incubation on ice, the samples were centrifuged at 13,860× g for 30 min at 4 °C, and the supernatants collected. Protein concentration in the supernatants was determined using the Bio-Rad Protein Assay Kit (Bio-Rad, Hercules, CA, USA). Aliquots were stored at −80 °C.