Hrp conjugated secondary antiserum

Proteins were separated using 10% SDS-PAGE and transferred to PVDF membrane. The membranes were probed overnight at 4°C with primary antibodies diluted in 5% milk/Tris-buffered saline with 0.1% Tween-20. Anti-GLT-1a antibody (generous donation from Paul Rosenberg, Harvard University) was used at a dilution of 1:80,000 and anti-xCT (Novus) was used at a 1:5,000 dilution. After incubation with HRP-conjugated secondary antiserum (Jackson Immuno; 1:10,000–1:50,000), immunoreactive bands on the membranes were detected by enhanced chemiluminescence (ECL Plus; GE Healthcare Bio-Sciences). Band density was measured using NIH ImageJ software. Blots were re-probed with anti-calnexin (Millipore, 1:40,000) as a loading control.

The PFC and NAc regions were dissected, homogenized in sucrose buffer containing protease inhibitors, and spun at 1000 g. The supernatant was retrieved and then spun at 10000 g. The resulting pellet was re-suspended in sucrose buffer and protease inhibitors, and spun at 10000 g. The supernatant was then discarded and the pellet containing the membrane fraction was suspended in 1% sodium dodecyl sulfate (SDS) in RIPA. Proteins were separated using 10% SDS-PAGE and transferred to PVDF membrane. The membranes were probed overnight at 4°C with primary antibodies diluted in 5% milk/Tris-buffered saline with 0.1% Tween 20. GLT-1 (1:5000, Chemicon, Billerica, MA) was measured in both the PFC and NAc, and GluA1 and GluA2 (1:1000, Chemicon, Billerica, MA) were measured in the NAc only. After incubation with HRP-conjugated secondary antiserum (Jackson Immuno; 1:10,000), immunoreactive bands on the membranes were detected by ECL Plus. Band density was measured using NIH Image J software.