Non fat dry milk protein

Brain cortical tissue, liver, mBCEC, or pBCEC were lysed by brief sonication in protein lysis buffer (containing 50 mM Tris, 10 mM EDTA, 1% Triton X 100, phosphatase inhibitor (PhosSTOP™), protease inhibitor cocktail (Roche Diagnostics cOmplete™), and pH adjusted to 7.5). Protein quantification was performed using bicinchoninic acid assay (BCA, ThermoFisher Scientific). Samples were mixed (4:1) with XT loading buffer (Bio-Rad) and proteins were denatured at 90 °C for 5 min. Proteins (20 μg/well) were loaded on NuPage® Novex 4–12% Bis-Tris Midi gels and separated under reducing conditions by SDS-PAGE using 1× MOPS or MES (Life Technologies) buffer. Then, proteins were electroblotted onto 0.45 μm polyvinylidene difluoride membranes (PVDF; GE Healthcare). Membranes were blocked with 5% non-fat dry milk protein (Bio-Rad) for 1 h at room temperature (RT). Membranes were incubated with primary antibody overnight in 5% bovine serum albumin (BSA; Roche) at 4 °C, followed by HRP-conjugated secondary antibody incubation in 5% non-fat dry milk protein for 1 h at RT. Detection of immune-reactive bands was performed using Clarity Western ECL Substrate (Biorad) and a ChemiDoc imager (Bio-Rad). Band intensities were determined using ImageLab software (version 5.2.1, Bio-Rad). All primary and secondary antibodies used are listed in Supplementary Table 1.