Ly111

ARSB activity measurements and determinations of sulfated glycosaminoglycans, and chondroitin-4-sulfate in the NCM460 and HT-29 cells were performed as previously reported [2 (link),3 (link)]. A fluorimetric assay was performed with the substrate 4-methylumbilliferyl sulfate (4-MUS) using 20 μl of cell homogenate and 80 μl of assay buffer (0.05M Na acetate buffer, pH 5.6) were combined with 100 μl of substrate (5mM 4-MUS in assay buffer) in wells of a microplate. The microplate was incubated for 30 minutes at 37°C, and the reaction was stopped by 150 μl of stop buffer (Glycine-Carbonate buffer, pH 10.7), and fluorescence was measured at 360 nm (excitation) and 465 nm (emission) in an microplate reader (FLUOstar, BMG LABTECH, Inc., Cary, NC).
The Blyscan™ assay kit (Biocolor Ltd, Newtownabbey, N. Ireland) was used for detection of the sulfated glycosaminoglycans (GAGs), based on the reaction of 1,9-dimethylmethylene blue with the sulfated oligosaccharides in the GAG chains. C4S content was determined following immunoprecipitation with C4S antibody (LY111, 0.5 μg/ml, Seikagaku, Tokyo, Japan). C6S antibody (0.5 μg/ml; LSBio, Seattle, WA) was used to detect C6S, and total CS-56 antibody (1:100; SCBT) to detect total CS, using previously reported methods [15 (link)].

NCM460 cells in which ARSB was silenced by siRNA, or treated by scrambled control siRNA, or untreated control cells were grown in 24-well plates. Sandwich ELISA was performed using C4S antibody (LY111, 0.5 μg/ml, Seikagaku, Tokyo, Japan) or C6S antibody (0.5 μg/ml; LSBio, Seattle, WA), with total CS-56 antibody (1:100; SCBT) as the coating antibody. Antibody signal intensity was compared for C4S and C6S antibodies, with signal intensity for CS-56 antibody considered as the signal for total CS.