Figure 6.

The centrosome recruitment of parkin, but not its dispersion, is dependent on the dynein motor protein. A, B, SH-SY5Y cells cotransfected with GFP (pseudocolored red) and siRNA for dynein heavy chain (DHC-siRNA) or control siRNA with scrambled sequence (Con-siRNA) were treated without (G) or with 5 μm MG132 for 12 h. Transfection of DHC-siRNA (B), but not Con-siRNA (A), markedly reduced MG132-induced centrosome accumulation of parkin (PKN, green). By itself, neither DHC-siRNA nor Con-siRNA had any significant effect on the subcellular distribution of parkin (G). Scale bar, 10 μm. Arrow, Parkin accumulation in transfected cells. C, D, After SH-SY5Y cells were treated with 5 μm MG132 for 24 h, they were incubated without MG132 (wash) for 24 h. At 12 h before the wash, cells were cotransfected with GFP and DHC-siRNA or Con-siRNA. Neither DHC-siRNA (D) nor Con-siRNA (C) had any significant effect on parkin dispersion at 24 h of wash. E, F, Transfection of DHC-siRNA (marked by GFP, arrow) greatly reduced the endogenous level of DHC as indicated by immunostaining (E) or Western blotting (F). The experiment was repeated three times with similar results. G, Statistical summary of the intensity of parkin accumulation under the conditions in (A–D). *p < 0.001 versus the preceding bar; n = 30 cells randomly selected from three independent experiments for each condition.