Anti glutaminase

Protein was isolated from cells using lysis buffer (20 mM Tris pH 8, 200 mM NaCl, 1 μm EDTA pH 8 0.5% Np40 and 10% glycerol). Lysates were standardized for protein content and 20 μg of total protein was separated by a gradient 4-20% SDS-polyacrylamide gel (Mini-PROTEAN TGX, Bio-Rad, CA, USA) electrophoresis and transferred to PVDF membranes using the iBlot system (Invitrogen, ThermoFisher Scientific, Grand Island, NY, USA). The membranes were blocked and primary antibody was diluted 1:1000 in 3% BSA in TBS containing 0.01% Tween 20 (TBST) and incubated overnight at 4°C. The following day, blots were washed in TBST buffer and incubated with goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (Dako, Carpinteria, CA, USA) for 1 hr at room temperature. After washing in TBST buffer (three times, 10 min per wash) the immunoreactive proteins were visualized using ECL detection reagent (Amersham, GE Healthcare Biosciences, Pittsburgh, PA, USA). The antibodies used were anti-ASCT2 (V501, Cell Signaling, Beverly, MA, USA) and anti-c-Myc (D84C12, Cell Signaling, Beverly, MA, USA), anti-glutaminase (Abcam, Cambridge, MA, USA) and anti-β-tubulin (Santa Cruz Biotechnology, Dallas, TX, USA).

Cells were washed with ice-cold PBS and incubated with RIPA buffer (Beyotime Institute Biotechnology, Shanghai, China) containing a proteinase inhibitor mixture (Roche, Mannheim, Germany) and 10 μM PMSF (Sigma, St. Louis, MO, USA). Lysates were briefly sonicated and cleared by centrifugation at 10,000 rpm for 20 min. Equivalent amounts of protein were analyzed using 10% SDS-PAGE gel electrophoresis. Proteins were transferred to polyvinylidene difluoride membranes and probed with antibodies. The following primary antibodies were used: anti-MAP2 (1:1000; Millipore Bioscience Research Reagents, Billerica, MA, USA), anti-PSD95 (postsynaptic density protein 95) (1:2000; Abcam, Cambridge, UK), anti-GluR1 (Glutamate Receptor 1) (1:300; Abcam), anti-MAOB (1:1000; Abcam), anti-GFAP (1:1000; Abcam), anti-GABA-T (1:1000; Abcam), anti-glutaminase (1:1000, Abcam), anti-β-actin (1:10000, Sigma). Secondary antibodies were HRP-conjugated anti-rabbit or anti-mouse antibodies (1:10000, Boster Bio-Technology, Wuhan, China). Visualization was performed using enhanced chemiluminescence (ECL, GE Healthcare Pharmacia). Densitometric analysis of Western blots was conducted using a ChemiDoc XRS (Bio-Rad, Hercules, CA, USA) and quantified using Quantity One version 4.1.0 (Bio-Rad).