Qpbca

Rat tissues or Bend.3 cells were homogenized by sonication in RIPA buffer. Protein concentrations were determined with a BCA assay kit (QPBCA, Thermo Scientific). Protein extracts were electrophoresed by 10% SDS-PAGE. The separated protein was then electrotransferred onto PVDF membranes, shaken slowly in blocking solution (5% milk in TBST) for 90 min and incubated overnight with antibodies against occludin (1:500, Invitrogen), claudin-5 (1:1000, Abcam), MAP3K2 (1:500, Invitrogen), p-JNK1/JNK2 (1:1000, Invitrogen), JNK (1:1000, Invitrogen), p-c-Jun (1:500, Invitrogen), c-Jun (1:500, Invitrogen), active caspase-3 (1:1000, Invitrogen), caspase-3 (1:2000, Invitrogen), and β-actin (1:5000, Millipore Sigma) as a loading control. Afterwards, the membranes were washed and transferred into a buffer with appropriate secondary antibodies for 1 h at room temperature. An ECL detection kit was used to visualize the protein bands. ImageJ (LOCI, Madison, WI, USA) software was used for quantitative analysis of western blot results.

The hippocampus from each hemisphere was dissected 12 h after HI. The tissues were immediately frozen on dry ice and stored at −80 °C. The tissues were then homogenized in a solution containing: Tris-HCl (25 mM, pH 7.9), NaCl (100 mM), ethylenediaminetetraacetic acid (5 mM), NP40 (1%), sodium butyrate (0.1 mM), Nam (5 mM), protease inhibitor (1%), phosphatase inhibitor, and phosphate buffered saline (PBS) (pH 7.4). Afterwards, samples were centrifuged 15,000g for 5 min at 4 °C. Protein concentration was measured by a BCA (QPBCA, Thermo Fisher Scientific, Waltham, MA, USA).