Phospho ser9 gsk3β

Brain regions were isolated 3 hr after the last escapable foot shock treatment, and western blotting was performed as described previously (Cheng et al., 2016 (link)). In brief, mouse hippocampus and prefrontal cortex were rapidly dissected in ice-cold phosphate-buffered saline, snap-frozen, and stored at −80° C before use. Brain regions were homogenized in ice-cold Triton-lysis buffer, and protein concentrations in the supernatants were determined using the Bradford protein assay. Proteins (10–20 μg) in brain region extracts were resolved with SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with antibodies to high mobility group box 1 protein (HMGB1; ab18256, Abcam), phospho-Ser9-GSK3β (#9322, Cell Signaling Technology), phospho-Ser21-GSK3α (#9316, Cell Signaling Technology), total GSK3α/β (05–412, Millipore), Rac1 (#ARC03, Cytoskeleton, Inc.), occludin (33–1500, Thermo Fisher Scientific), claudin-5 (35–2500, Thermo Fisher Scientific), ZO-1 (40–2200, Thermo Fisher Scientific), ZO-2 (71–1400, Thermo Fisher Scientific) and reblotted with β-actin (Sigma Aldrich) as the loading control. Chemiluminescent signals were acquired using an Amersham Imager 600 (GE Healthcare Life science), which determines the most high resolution digital image. The images were quantified using the IQTL software (GE Healthcare Life science).

The hippocampus was rapidly dissected in ice-cold phosphate-buffered saline and stored at −80°C after being snap-frozen. Brain regions were homogenized in Triton lysis buffer containing 20 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton-100, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 5 μg/ml pepstatin, 1 mM phenylmethanesulfonyl fluoride, 1 mM sodium vanadate, 50 mM sodium fluoride, and 100 nM okadaic acid. Tissue lysates were centrifuged at 14000 rpm for 10 min at 4°C to remove the insoluble fraction. The Bradford protein assay was used to determine the concentration of protein in the supernatants. Hippocampal proteins (5-20 μg) were resolved with SDS-PAGE, transferred to nitrocellulose membranes and then immunoblotted. Antibodies used were to phospho-Ser9-GSK3β (#9336, Cell Signaling Technology), total GSK3β (#610202, BD Transduction Laboratories), phospho-Ser21-GSK3α (#9316, Cell Signaling Technology) and total GSK3α/β (clone 4G-1E, #05-412, Millipore). The membranes were reblotted with β-actin (#A5441, Sigma Aldrich) to ensure equal protein loading.

Mouse hippocampi were rapidly dissected in ice-cold phosphate-buffered saline. Brain regions were homogenized in ice-cold lysis buffer containing 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 10% glycerol, 1 μg/ml leupeptin, 1 μg/ml aprotinin, 1 μg/ml pepstatin, 1 mM phenylmethanesulfonyl fluoride, 50 mM NaF, 1 mM sodium orthovanadate, and 100 nM okadaic acid. The lysates were centrifuged at 20,800xg for 10 min. Protein concentrations in the supernatants were determined using the Bradford protein assay (Bradford 1976 (link)). Lysates were mixed with Laemmli sample buffer (2% SDS) and placed in a boiling water bath for 5 min. Proteins (10 μg) were resolved in SDS-polyacrylamide gels, transferred to nitrocellulose, and incubated with primary antibodies to phospho-Ser21-GSK3α (#9316L; Cell Signaling Technology), phospho-Ser9-GSK3β (#9336; Cell Signaling Technology) and total GSK3α/β (#05-412; Millipore). Immunoblots were developed using horseradish peroxidase-conjugated goat anti-mouse, or goat anti-rabbit IgG, followed by detection with enhanced chemiluminescence.

Proteins (5–20 μg) in brain region extracts prepared as described above were resolved with SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with antibodies to HMGB1 (ab18256, Abcam), IκBα (#9242, Cell Signaling Technology), active NF-κB (MAB3026, Millipore), NLRP3 (AG-20B-0014, Adipogen), active p20 caspase-1 (AG-20B-0042, Adipogen), phospho-Ser9-GSK3β (#9322, Cell Signaling Technology), phospho-Ser21-GSK3α (#9316, Cell Signaling Technology), total GSK3α/β (05–412, Millipore), and reblotted with β-actin (Sigma Aldrich) to ensure loading consistency.