Brain Proteoglycan Extraction and Analysis

Extraction of PGs from the brain was carried out as described previously [26 (link)]. The brain lysates (600 μg of proteins) were digested with 5 milliunits of chondroitinase ABC (EC4.2.2.4; Seikagaku Corporation, Tokyo, Japan) and 5 units of β-HexNAcase for 2 h at 37 °C. Undigested and chondroitinase ABC-digested lysates (40 μg of proteins) were separated by Bullet PAGE One Precast Gel 5–20% (Nacalai Tesque Inc., Kyoto, Japan). Immunoblotting was performed using the following primary antibodies, as described previously [16 (link)]: anti-aggrecan antibody (dilution: 1/2000; AB1031, Merck Millipore, Burlington, MA, USA), anti-brevican antibody (Cat. No. RUO-610894, BD Biosciences, San Jose, CA, USA), anti-phosphacan antibody (dilution: 1/200; clone 3F8, DHSB, Iowa, IA, USA), Cat316 (dilution:1/10,000), 1B5 (dilution: 1/10,000; Cat. No. 270431, Sekagaku Business Corp., Tokyo, Japan), and 2B6 (dilution: 1/10,000; Cat. No. 270432, Sekagaku Business Corp., Tokyo, Japan).