Hrp conjugated anti rabbit or anti mouse antibodies

Homogenate aliquots (30 μg of total protein) were boiled and then electrophoresed in 10% SDS-PAGE acrylamide gels, transferred onto nitrocellulose membranes (Bio-Rad) and incubated for 1 h in Tris-buffered saline and 0.1% Tween 20 (TBS-T) containing 5% nonfat milk. Membranes were then incubated overnight at 4°C with primary antibodies against occludin (1:300, Invitrogen), HIF-1α (1:300, Novus), VEGF (1:500, Abcam), washed in TBS-T, and then incubated for 2 h at room temperature with corresponding HRP-conjugated anti-rabbit or anti-mouse antibodies (1:3000, Boster). The membranes were developed with the SuperSignal West Pico HRP substrate kit (Pierce) and photographed on a Gel DOC^TM XR⁺ image station (Bio-rad). Protein band intensities were quantitated after normalization to β-actin or total protein stained by Ponceau S.

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).

Homogenate aliquots (50 μg of total protein) were boiled and then electrophoresed in 12% SDS‐PAGE acrylamide gels, transferred onto PVDF membrane (Millipore, Billerica, MA, USA), and incubated for 1 h in TBS‐T (Tris‐buffered saline and 0.1% Tween‐20) containing 5% nonfat milk. Membranes were then incubated with primary antibodies against p‐AMPK (1:1000; Cell Signaling Technology, Danvers, MA, USA), AMPK (1:1000; Cell Signaling Technology), gp91^phox (1:1000; BD Transduction Laboratories, Lexington, KY, USA), occludin (1:300; Invitrogen), or claudin‐5 (1:500; Invitrogen) overnight at 4°C, washed in TBS‐T, and then incubated for 1 h at room temperature with corresponding HRP‐conjugated anti‐rabbit or anti‐mouse antibodies (1:2000; Boster). The membranes were developed with the SuperSignal West Pico HRP substrate kit (Pierce, Rockford, IL, USA) and photographed. Protein band intensities were quantitated after normalization to beta actin.