i.c.v. administration to SD mice.

A 1–5 mg/kg BW dose of modB or mod2B, or the same volume of PBS, was injected into 10-week-old SD mice by i.c.v. administration (5). For repeated administration (twice) at 2-week intervals beginning from the age of 10 weeks, the enzyme solutions were injected at the same site in the same manner. One week after the injection, the SD mice were sacrificed, and their brains, livers, and spleens were dissected after PBS perfusion. Each tissue was frozen immediately. The sections were thawed and homogenized by sonication in PBS containing protease inhibitors. After centrifugation at 13,200 g, the supernatants were collected as tissue extracts. The MUGS-degrading activities per wet weight of tissues were calculated.

For immunoprecipitation (IP), protein A-agarose (Bio-Rad) was washed with 1% (v/v) Triton X-100 (Nacalai Tesque)/TBS (25 mM Tris [Sigma-Aldrich], 137 mM NaCl [Nacalai Tesque], 2.7 mM KCl [Sigma-Aldrich]; pH 7.4). After binding with anti-NAG(A) specific for human HexA at 4°C overnight, the brain extracts were treated at r.t. for 2 hours. After washing, the bound Hex proteins were eluted with SDS sample buffer containing 50 mM Tris-HCl (Wako) (pH 6.8), 6% (v/v) glycerol (Wako), 0.2% (v/v) 2-ME (Wako), 4% (w/v) SDS (Sigma-Aldrich), and 0.002% (w/v) bromophenol blue (Kishida Chemical). Each sample was subjected to SDS-PAGE and immunoblotting. The PVDF membranes were treated with human HexA-specific Ab (1:1,000 dilution) and Clean-Blot IP Detection solution (Thermo Scientific, 1:500 dilution). The molecular weight of each recombinant Hex was calculated based on that of the Biotinylated Protein Ladder (Cell Signaling Technology). The Image J software program (version 1.46) (61) was used to quantify the signal intensity.

For IHC, each brain hemisphere was put in O.C.T. compound (Sakura Seiki) at –20°C. Slices (10-μm thick) were prepared with a Microm HM550 cryostat (Microedge Instruments), which were then placed on silanized slides (Dako). Each slice was treated with human HexA-specific Ab (1:2,000 dilution). The specimens were viewed with a BIOREVO BZ-9000.