Appropriate secondary antibodies

Brain tissues were homogenized in an ice-cold immunoprecipitation buffer (IP buffer) (containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM EDTA, 10% glycerol, 1% Nonidet p-40, phosphatase inhibitor cocktail 2 (Sigma) and protease inhibitor cocktail (Merck)). The homogenate was centrifuged at 15,000g for 30 min at 4 °C and the supernatant was determined using a coomassie protein assay reagent (Thermo). Equal amounts of extracted proteins were respectively loaded on 4–12% NuPAGE Bis-Tris Gels (Thermo) and subjected to immunoblotting. The following primary antibodies were used: rabbit anti-ZO1 (1:500, Thermo); mouse anti-occludin (1:2000, Thermo); rabbit PKC-δ (1:500, cell signaling); mouse anti-GAPDH (1:5000, Proteintech). Appropriate secondary antibodies (GE Healthcare) were incubated with the membranes for 1 h at room temperature. For Co-Immunoprecipitation assay, immunoprecipitated proteins were detected using VeriBlot for IP Detection Reagents (Abcam) without being contaminated by IgG heavy and light chains from the precipitated-antibodies. The signals were visualized using Amersham™ ECL Select Western Blotting Detection Reagent (GE Healthcare) according to the manufacturer’s instructions and recorded via UVP BioSpectrum Image system (UVP). Data were analyzed using Vision Works LS software (UVP).