Figure 2

Parkin mediates degradation of mutant GCase. (A) Skin fibroblasts from a GD patient or normal skin fibroblasts were transfected with wt or mutant myc-parkin (T240R). Forty-eight hours later cell lysates were prepared and subjected to western blot analysis. The corresponding blot was interacted with anti-GCase, anti-myc and anti-actin antibodies, as a loading control. (B) To normalize the results, the blots were scanned and GCase intensity at each lane was divided by the intensity of actin. The value obtained for non-transfected cells was considered as 1. The results represent the mean ± SEM, of three independent experiments. *P < 0.02, **P < 0.01 in Student t-test. (C) Skin fibroblasts from a normal individual or from a GD patient were transfected with shRNA containing plasmid designed to down-regulate parkin expression (shParkin) and pLKO.1 plasmid harbouring shRNA against GFP as a control (control). Forty-eight hours later, cell lysates were prepared and subjected to western blot analysis. The corresponding blot was interacted with anti-parkin, anti-GCase, and anti-ERK antibodies as a loading control. To quantify the results, the blots were scanned and GCase or parkin intensity at each lane was divided by the intensity of ERK1/2 in the same lane. The value obtained for control (in normal or GD cells) was considered as 1. The numbers appear below the blots. The cells used in this experiment derived from patient no. 3 and normal individual no. 1 (see Table 1).