Pcamkii t286

Western blots were performed using antibodies against NFAT1(Cell
Signalling, 5861S), pS6K(T389) (Cell Signaling, 9234S), pCamKII(T286) (Cell
Signalling, 12716S), MeCP2 (Cell Signalling, 3456S), pMeCP2(S421)(Rockland,
600-401-X14), Firefly luciferase (Thermo, PA5-32209). The Electrophoresis
and Membrane-Transfer were performed by iBlot (Invitrogen). Imaging was
obtained by Odyssey (Li-Cor) system, following the manufacturer’s
protocol.

NAc tissues were homogenized in a solution containing 50 mM Tris buffer (pH 7.4), 2 mM EDTA, 5 mM EGTA, 0.1% SDS, protease inhibitor cocktail (Roche, Indianapolis, IN), and phosphatase inhibitor cocktail type I and II (Sigma). Homogenates were centrifuged at 500 g for 15 min and supernatants were collected. Proteins were analyzed using Bradford protein assay (BioRad, Hercules, CA). Proteins were separated by PROTEAN TGX gels at 100 V for 1 h, transferred onto PVDF membranes at 30 V for 1 h (BioRad, Hercules, CA), and incubated with antibodies against pCaMKII (T286) (Cell Signaling, Danvers; 9102; 1:500), CaMKII (Cell Signaling, Danvers; 9102; 1:500), pNR2B (S1303) (Cell Signaling, Danvers; 9102; 1:500), pGluR1(S831) (Cell Signaling, Danvers; 9102; 1:500), pPKCγ(T514) (Cell Signaling, Danvers; 9102; 1:500), PKCγ; (Cell Signaling, Danvers; 9102; 1:500), pNR1(S896) (Cell Signaling, Danvers; 9102; 1:500), Ng (Cell Signaling, Danvers; 9102; 1:500), pAKT(S473) (Cell Signaling, Danvers; 9102; 1:500), AKT (Cell Signaling, Danvers; 9102; 1:500), mGluR5 (Cell Signaling, Danvers; 9102; 1:500) and GAPDH (Millipore, Burlington, MA; MAB374; 1:2000). Chemiluminescent bands were detected on an Image Station and quantified using NIH Image J software.