Pierce ecl western blotting detection kit

Cells were collected in 1× RIPA buffer containing 1 mM PMSF and sonicated for 10 s. Protein concentrations were measured using the bicinchoninic acid (BCA) assay (Pierce). Equal amounts of protein (20 μg/lane) were loaded onto 4%–15% precast protein gels (Bio-Rad) and transferred to polyvinylidene difluoride (PVDF, Bio-Rad) membranes. Membranes were blocked for 1 hr with TBS/0.1%-Tween buffer plus 5% (w/v) non-fat dried milk and incubated overnight at 4°C with primary antibodies dissolved in blocking buffer. Membranes were incu-bated with secondary antibodies for 1 hr and developed using a Pierce^® ECL Western blotting detection kit (Thermo Scientific) and a ChemiDoc XRS System (Bio-Rad). Image lab software was used to quantitate Western blot signals. The following primary antibodies were used in this study: mouse anti-CaMKK β (1:200; Santa Cruz Biotechnology), rabbit anti-SIRT1 (1:1000; Cell Signaling), rabbit anti-phospho-SIRT1 (1:1000; Cell Signaling), rabbit anti-phospho-CAMKIV (1:1000; Abcam), rabbit anti-eNOS (1:1000; Cell Signaling), rabbit anti-VCAM-1 (1:1000; Cell Signaling), rabbit anti-CD54/ICAM1 (1:1000;Cell Signaling) and mouse anti-β-actin (1:5000; Sigma-Aldrich). HRP (horseradish peroxidase)-conjugated anti-rabbit IgG (1:2000; Vector Laboratories) and HRP-conjugated anti-mouse IgG (1:2000; Vector Laboratories) were used as secondary antibodies.

Total protein from the cerebral cortex or mBMEC culture was homogenized in lysis buffer and isolated as described (19 (link)). The blots were blocked with 5% non-fat milk and incubated with mouse anti-Claudin5 antibody (Thermo Fisher Scientific, Cat #35–2500) or β-actin (Sigma-Aldrich) for 1 h at room temperature. Then membranes were incubated with HRP-conjugated secondary antibodies (Cell Signaling) for 1 h and developed using a Pierce® ECL Western blotting detection kit (Thermo Scientific) and Amersham High-Performance Chemiluminescence Films (GE Healthcare). ImageJ software was used to quantify western blot signals. The relative claudin-5 amount was corrected by β-actin and then normalized by WT sham or non-OGD controls.