Anti phospho gsk3

Cells were lysed using complete radioimmune precipitation assay (RIPA) buffer supplemented with complete ULTRA protease inhibitor cocktail tablets (Roche, Basel, Switzerland) and sodium orthovanadate. Anti-Akt2, anti-phospho-Akt (Thr308), anti-phospho-Akt (Ser473), anti-Pras40, anti-phospho-Pras40 (Thr246), anti-Gsk3A, anti-phospho-Gsk3 (Ser21/9), anti-Tsc2, anti-phospho-Tsc2 (Thr1426), anti-mTOR, anti-phospho-mTOR (Ser2448), anti-p70S6k1, anti-phospho-p70S6k1 (Thr389), anti-p70S6K2, anti-4E-BP1, anti-4E-BP1 (Thr37/46) (Cell Signalling Technology, Danver, Massachusetts, USA) rabbit polyclonal antibodies were used in immunoblotting. Luciferase constructs (pLightSwitch_3′UTR) (Switchgear genomics, Carlsbad, CA, USA) containing the 3′ UTR region of Akt2, Pras40, Gsk3a, CSF1 and Itgb1 was individually transfected into HEK293T cells using pGL4.12 (luc2CP) as a normaliser. Luciferase activity was measured by using the dual luciferase assay (Promega).

Tissue corresponding to the NAc or CPu was collected from brains of each male or female rat (N = 6–9) rapidly after sacrifice. Following homogenization, 30–50 μg of protein from the indicated region were incubated in sample buffer for 3–5 min at 95 °C. Proteins were resolved by electrophoresis on 10% polyacrylamide gels under denaturing conditions (SDS–PAGE) and transferred onto nitrocellulose or PVDF membranes (Bio-Rad Laboratories, Hercules, CA) using a semidry transfer system (Invitrogen). Membranes were blocked in TBS-Tween (TBS-T)/5% nonfat milk for 1 h followed by incubation with PBS-T/5% nonfat milk containing the indicated first antibody overnight at 4 °C. Membranes were washed in TBS-T (3 × 10 min) and incubated with the appropriate horseradish peroxidase (HRP)-conjugated polyclonal secondary antibody (Bio-Rad) for 2 h at room temperature. After three washes as indicated above, signal detection was performed using a chemiluminescence kit (Perkin-Elmer). The primary antibodies used were anti-phosphoGSK-3 (1:1000, Cell Signaling), anti-BDNF (1:1000, Abcam), anti-phosphoTrkB (1:1000, Cell Signaling), and rabbit anti-GAPDH (1:10,000 or 1:20,000 Abcam).

Protein extracts were prepared and western blot analyses performed as described^{60 (link)}. Briefly, equal amount of proteins was resolved by 10% Novex Bis-Tris Gels (Invitrogen) and blotted onto nitrocellulose membranes. Membranes were blocked (5% non-fat dry milk (DM) or 5% BSA in Tris-buffered saline (TBS: 10 mM Tris-HCl (pH 7.5), 150 mM NaCl) with 0.1% Tween, 1 h, RT) and incubated with primary antibodies diluted in 5% milk/TBS/T and/or 5%BSA/TBS/T (overnight, 4 °C): rabbit polyclonal anti-Cyclin D2 (1:1 000, sc-593), anti-PPARδ (1:500, sc-7197) (both Santa Cruz, DM), anti-phospho-GSK-3 (DM, 9336), anti-pan-actin (DM, 4968), anti-PDK1 (BSA, 3062) (all 1:1 000, Cell Signaling), rabbit monoclonal anti-β-catenin (1:1 000, BSA, 9582), anti-GSK-3β (1:1 000, DM, 9315), mouse monoclonal phospho (308) anti-Akt (1:1 000, BSA, 9275), anti-Akt (1:1 000, DM, 2920) (all Cell Signaling), anti-KIP/p27 (1:2 500, DM, 610241), anti-PARP (1:1 000, DM, 611038) (all BD Transduction Laboratories). Antigen/antibody complexes were visualized using horseradish peroxidase-conjugated secondary antibodies (Amersham) and Super Signal@ ECL detection system (Bio-Rad).