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  • Proteins were separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) for 55–65 min at 150 V, and transferred to Immobilon-P transfer membranes (Cat # IPVH00010, 0.45 µm pore size; Millipore Co., Bedford, MA) in transfer buffer (50 mM Tris, 250 mM glycine, 3.5 mM SDS with 20% methanol) using a Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad Laboratories Ltd., Hercules, CA) for 110 min at 75 V. Transfer membranes were then incubated with blocking buffer (5% dry milk powder in PBS containing 0.5% Tween 20) for 1 h at room temperature and then incubated overnight at 4°C in blocking buffer with primary antibodies against the following proteins: DARPP32 (1∶2000, 374-DARPP, PhosphoSolutions, Aurora, CO), pDARPP32 Thr34 (1∶1000, p1025–34, PhosphoSolutions, Aurora, CO), pDARPP32 Thr75 (1∶1000, p1025–75, PhosphoSolutions, Aurora, CO), CREB (1∶1000, 9104, Cell Signaling, Danvers, MA), pCREB (1∶1000, 9196L, Cell Signaling, Danvers, MA), respectively.
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