CHO-K1 (ATCC CCL-61) and mutant CHO cell lines PgsA-745 (ATCC CRL-2242), PgsB-618 (ATCC CRL-2241), PgsC-605 (ATCC CRL-2245), PgsD-677 (ATCC CRL-2244), and PgsE-606 (ATCC CRL-2246) were obtained from the American Type Culture Collection. The prostate cancer cell line DU145 was kindly provided by Professor Shoko Nishihara, Soka University. The cells were cultured in RPMI 1640 medium (Gibco, Tokyo, Japan) supplemented with 10% v/v FBS (Gibco) or CSS (Gibco), 100 U/ml penicillin G, and 100 µg/ml streptomycin. Purified chondroitin sulfate A (CSA) from whale cartilage, chondroitin sulfate C (CSC) from shark cartilage, dermatan sulfate (DS) from porcine skin, keratan sulfate (KS) from porcine nasal cartilage, and heparan sulfate (HS) from porcine kidney were purchased from PG research (NaCS-A2, NACS-C2, NADS-B2, NSKS2, and NaHS-P2, Tokyo, Japan, http://www.pg-r.com). Hyaluronic acid (HA) from Streptococcus zooepidemicus was provided by Shiseido Co. (Tokyo, Japan). Chemically modified 2-O-ΔS-heparin, 6-O-ΔS-heparin, N-ΔS-heparin, and purified HS were obtained from Iduron (DSH001/2, DSH002/6, and DSH004/NAc, Manchester, U.K.). Purified chondroitin sulfate D (CSD) from shark cartilage (NaCS-D2(Shc)) and biotinylated heparin from porcine intestine (BHHep-Na(PgI)) were obtained from PG Research. Purified chondroitin sulfate E (CSE) from squid cartilage (034-23061) was obtained from Wako Pure Chemicals (Tokyo, Japan). GAGs except for heparin were biotinylated via carboxyl or aldehyde groups by cross-linking. Reaction solutions [1 mg/ml GAG in 100 mM MES-NaOH (pH 5.3), 1.25 mM EZ-link hydrazide biotin (Thermo Fisher Scientific, Tokyo, Japan) and 125 µg/ml 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (Thermo Fisher Scientific)] were incubated at 20°C for 12 h in the dark. The solution was dialyzed against PBS.
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