Fig. 3.

The highly conserved R99 is critical for ARSG activity. (A) In HEK293T cells, similar overexpression levels of C-ter Myc-tagged WT (WT-Myc) or variant (p.R99H-Myc) human ARSG are validated in a Western blot assay (WB) by their specific immunoreactivity with an anti-Myc antibody (asterisk). Proteins are detected at the expected molecular weight (63 kDa). NT, nontransfected cells. Adjusted levels of total proteins are confirmed by levels of the housekeeping calnexin. (B) These HEK293T cells were used to measure ARSG activity, expressed in nmol/h/mg of protein, using a method adapted from Frese et al. (32). Vertical bars represent the SEM for seven experiments. (C) Total leukocyte arylsulfatase activity was compared between homozygous (G/G) healthy controls, taken as the 100% reference activity level, and homozygous (A/A) affected ASTs. The number of sampled dogs is identified within each histogram, and vertical bars represent SEM.