Ecl detection reagent

Under deep anesthesia (sodium pentobarbital i.p.; 50 mg/kg), rats were euthanized at 24 h. The striatal region of the brain was rapidly isolated and homogenized, and total proteins were extracted using protein extraction buffer (Mammalian Cell-PE LBTM, Geno Technology, USA) containing protease and phosphatase inhibitors (Complete Mini, Roche Diagnostics, Indianapolis, IN, USA). Lysed tissues were centrifuged at 12,500 rpm for 15 min (at 4 °C), and the supernatant was collected for storage at −80 °C or to perform a Western blot analysis. Proteins were separated by gel electrophoresis, and then electroblotted onto polyvinylidene difluoride (PVDF) membranes (PerkinElmer Life Sciences, USA). Membranes were blocked with 5% non-fat milk, and then incubated overnight at 4 °C with the indicated antibodies, including heme oxygenase (HO)-1, Bax, cleaved caspase-3, Beclin-1, LC3-I/II, and p62 (1:10³ dilution) followed by an appropriate secondary antibody (1:2 × 10⁴ dilution) for 1 h at room temperature. Signals were visualized using enhanced chemiluminescent (ECL) detection reagents (PerkinElmer Life Sciences). The membrane was then stripped and reprobed with an antibody specific for β-actin (1:10⁴ dilution) to ensure the accuracy of each loading. Protein expression was quantified by a BioImaging System (Level Biotechnology).

All chemicals were purchased from Sigma (St. Louis, MO) and antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA), respectively, unless specified otherwise. Antibody for HBP1 was from Novus Biologicals (Littleton, CO), and α-tubulin was from Abcam (Cambridge, MA). PVDF membranes and ECL detection reagents for Western blotting analysis were purchased from Perkin Elmer Life Sciences, Inc. (Waltham, MA). Dual-light^® system was from Applied Biosystems (Foster City, CA). PI3K inhibitor LY 294002 was purchased from Sigma (St. Louis, MO). Small interfering RNA (siRNA) molecules specific for HBP1 was purchased from Invitrogen (Carlsbad, CA).

For Western blotting analysis, the cells were lysed on ice for 30 min in a lysis buffer containing fresh protease and phosphotase inhibitor cocktails (Sigma). The lysates were then centrifuged (12000 g at 4 °C for 15 min). Protein concentrations were determined by protein assay (Bio-Rad, Hercules, CA). Equivalent proteins were denatured in protein loading buffer, loaded onto 10% SDS-PAGE gels, and subsequently transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA) by electroblotting. The PVDF membranes were blotted with 5% nonfat milk in TBST buffer (Bio-Rad) for 1 h and incubated overnight at 4°C with antibodies against galectin-1, COX2, β-actin (Abcam) and AKT, p-AKT, mTOR, p-mTOR (CST). Signals were detected using ECL detection reagents (Perkin-Elmer Life Sciences, Boston, USA) following the manufacturer's instructions.