Figure 1

Mutant GCase undergoes ubiquitination and associates with parkin. (A) Protein lysates were prepared from MG-132-treated GD-derived skin fibroblasts or normal skin fibroblasts under denaturing conditions. GCase was immunoprecipitated with monoclonal anti-GCase antibody and subjected to western blot analysis and interaction with polyclonal anti-GCase antibodies. (B) Densitometry was used to quantify the level of ubiquitination. To normalize the results, intensity of ubiquitinated GCase in each lane was divided by the intensity of total GCase in the same lane. The value obtained for normal cells was considered as 1. The results represent the mean ± SEM of 3–6 independent experiments. ***P < 0.001 in Student t-test. (C) Lysates were prepared from MG-132-treated GD-derived skin fibroblasts or from normal skin fibroblasts. They were subjected to western blot analysis and interaction with anti-GCase antibody, anti-parkin antibody, or anti-actin antibody as a loading control. (D) Parkin-containing complexes were immunoprecipitated from lysates of MG-132-treated GD-derived skin fibroblasts or normal skin fibroblasts with anti-parkin antibody. They were subjected to western blot analysis and interaction with anti-GCase antibody. (E) For loading control, 5% of lysates were subjected to SDS-PAGE and western blot analysis with anti-GCase or anti-parkin antibodies. Patients’ details appear in Table 1. Numbers in parenthesis denote patient-derived cell lines, shown in Table 1.