Immunoblot analysis was carried out as a previously described method [48 (link)]. Samples from right cerebral cortexes were homogenized in lysis buffer [1 % Triton X-100 and 1 mM EDTA in PBS (pH 7.4)] using a tissue homogenizer and centrifuged at 16,000 g for 20 min at 4°C. Supernatant was isolated and protein concentration of each sample was calculated with bicinchroninic acid (BCA) protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s manual. Protein (30 µg) from each sample was loaded on 10 % sodium dodecyl sulfate poly-acrylamide gel electrophoresis and electrophoresed until dye went down to the bottom. Protein was transferred to a poly-vinylidene fluoride membrane and membranes were incubated with 5 % skim milk in tris-buffer saline containing 0.1 % Tween-20 (TBST) for 1 h. Membranes were washed three times with TBST for 10 min and reacted with following primary antibodies: anti-parvalbumin (1:1,000; Thermo Fisher Scientific) and anti-β-actin (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4°C. They were washed with TBST and incubated with horseradish peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG (1:5,000; Thermo Fisher Scientific) for 2 h. Membranes were washed with TBST and reacted with enhanced chemiluminescence Western detection reagents (GE Healthcare) according to the manufacturer’s manual.
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