Fig 2: Western blot of patient fibroblast cell lysates. An immunoreactive 56kDa band was observed in samples from control cells and from slowly progressing mucopolysaccharidosis type IIIA patient cells cultured at 30 °C. ß‐actin was used as a protein loading control. Culture temperatures in °C are indicated below the lanes. SGSH = sulfamidase.

Fig 3: Linearity of (A) protein concentration and (B) incubation time in sulfamidase (SGSH) activity assay in cultured fibroblasts of control cell line.

Fig 4: Residual sulfamidase (SGSH) enzyme activity in fibroblasts at 37 °C and at 30 °C. Group 1 (n = 23) was homozygous or compound heterozygous for the common severe mutations p.R245H, p.Q380R, p.S66W, or c.1080delC. Group 2 (n = 18) was compound heterozygous for 1 of the severe mutations and the mild p.S298P mutation. Group 3 (n = 4) was homozygous for the mild p.S298P mutation. The limit of quantification is indicated as a dashed line.

Fig 5: Western blot (A) and sulfamidase (SGSH) enzyme activity (B) in fibroblast cell lysate with addition of bortezomib at 2 concentrations (0 and 10nM) for 48 hours. Mucopolysaccharidosis type IIIA (lane 2: c.1080delC/c.376insG; lanes 3–6: p.S298P/p.S298P) and control fibroblasts (lane 1) were cultured at 37 °C and 30 °C for 48 hours instead of 1 week. To show the location of the mature SGSH band more accurately a diluted control sample was loaded in lane 7. SGSH activity was measured in p.S298P/p.S298P cell lysates.