Protein concentration in hippocampal extracts from mice and human tissue was
measured using the BCA Protein Assay Kit (Cole-Parmer). Forty micrograms of
protein/lane were electrophoretically separated on a 12% SDS polyacrylamide gel
and electrically transferred onto a Hybond-P PVDF transfer membrane (Millipore)
for 1 h. Membranes were blocked in PBS-milk 5% at RT for 1 h. Next, membranes
were incubated in block solution overnight with the following primary
antibodies: mouse anti-glutamine synthetase (1:500; Abcam), mouse anti-GAPDH
(1:1,000; Abcam), rabbit anti-Cyclophilin B (1:1,000; Sigma) and rabbit
anti-β-actin (1:1,000; Abcam). Membranes were incubated for 1 h with IRDye 680CW
goat anti-mouse antibody, IRDye 800CW goat anti-mouse antibody, or IRDye 800CW
goat anti-rabbit antibody (LI-COR, 1:20,000), then scanned with an Odyssey
infrared imaging system (LI-COR) and analyzed using Un-Scan-It gel version 6.1
(Silk Scientific).
measured using the BCA Protein Assay Kit (Cole-Parmer). Forty micrograms of
protein/lane were electrophoretically separated on a 12% SDS polyacrylamide gel
and electrically transferred onto a Hybond-P PVDF transfer membrane (Millipore)
for 1 h. Membranes were blocked in PBS-milk 5% at RT for 1 h. Next, membranes
were incubated in block solution overnight with the following primary
antibodies: mouse anti-glutamine synthetase (1:500; Abcam), mouse anti-GAPDH
(1:1,000; Abcam), rabbit anti-Cyclophilin B (1:1,000; Sigma) and rabbit
anti-β-actin (1:1,000; Abcam). Membranes were incubated for 1 h with IRDye 680CW
goat anti-mouse antibody, IRDye 800CW goat anti-mouse antibody, or IRDye 800CW
goat anti-rabbit antibody (LI-COR, 1:20,000), then scanned with an Odyssey
infrared imaging system (LI-COR) and analyzed using Un-Scan-It gel version 6.1
(Silk Scientific).