Western blotting. Aliquots of isolated mitochondria and brain homogenates were subjected to freeze- thaw cycles and sonication. Equal amounts of proteins were heated in reducing sample buffer [191] and then loaded onto 1.50 mm thick polyacrylamide mini gels (12%) and separated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mini-PROTEAN® 3 Cell; Bio-Rad Laboratories, Inc., Hercules, CA, USA). Prestained protein markers (New England BioLabs Inc., Ipswich, MA, USA) were used as size standards in the electrophoresis. Proteins were transferred at 350mA for 3 hours (Mini Trans-Blot® Electrophoretic Transfer Cell; Bio-Rad Laboratories, Inc.) to 0.45 µm nitrocellulose membranes (Amersham Hybond®-ECL; GE Healthcare Life Science). Ponceau-S staining of membranes was performed to check the efficiency of proteins transfer. For immunoblotting, membranes were blocked for 2 hours at room temperature with 5% fatty acid/globulin-free bovine serum albumin (Sigma-Xxxxxxx Co.) or 5% skimmed milk, depending on the antibody, in phosphate-buffered saline (pH 7.4) with 0.1 % Tween® 20 (PBST). Then membranes were incubated overnight at 4°C in the primary antibody with PBST. The primary antibodies used to evaluate protein expression are: • Disulfide Isomerase (PDI) at 1:1000 (Cell Signaling); • Histone H3 at 1:2000 (Cell Signaling); • GRP75 at1:1000 (Cell Signaling); • PYK2 at 1:1000 (Bd Transduction); • Paxillin at 1:1000 (Bd Transduction). After overnight incubation in primary antibody, the membranes were rinsed three times in PBST and incubated in secondary antibody for one hour r.t. in HRP- conjugated goat anti-rabbit IgG (1:5000) or anti-mouse IgG (1:3000). The blots were rinsed thoroughly in PBST and were developed using Xxxxxx XxxxxXxxxxx West Pico chemiluminescent substrate and then exposed to CL-XPosure film (Thermo Scientific).
