Figure 2.

Temporal Analysis of WT SGSH and SGSHv4 Activity

HEK293 cells were transfected with vectors expressing WT SGSH or SGSHv4, the (A) media and (B and C) cells harvested at the indicated time points, and SGSH activity (A and B) or protein levels (C) assessed; n = 3 biological replicates, in triplicate. Data represent mean ± SD. (D) The ratio of WT SGSH to SGSHv4 protein levels in the cell lysate as assessed by western blot. Both precursor and mature forms were evaluated; n = 3 biological replicates. Data represent mean ± SD. (E) Representative western blot (of four biological replicates) indicating SGSH and SGSHv4 protein levels in the indicated fractions isolated after gradient centrifugation. LAMP1, Bip, and Grasp 65 were used to identify enrichment of lysosomal, ER, and Golgi markers, respectively. Asterisk (*) indicates unglycosylated SGSH; arrowhead indicates glycosylated SGSH. PNF, post-nuclear fraction.