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Fig. 4. Lactacystin-induced recruitment of transfected parkin in HEK293 cells. HEK293 cells were transfected with a parkin expression construct, treated without (-Lac; A,C,E) or with (+Lac; B,D,F) 10 µM lactacystin for 12 hours, and co-stained with antibodies against {gamma}-tubulin ({gamma}-tub; A,B) and parkin (PKN; C,D). Merged confocal images (E,F) show that parkin accumulated in the centrosome in some of the transfected cells after lactacystin treatment (indicated by arrows in F). Scale bars: 5 µm. (G) Percentage of transfected cells that have centrosomal accumulation of parkin was plotted against the concentration of the lactacystin applied (12 hours). (H) Percentage of transfected cells that have centrosomal accumulation of parkin was plotted against the duration of lactacystin treatment (10 µM). *P<0.05 vs. 0 µM (G) or 0 hour (H) (n=3 fields of view per data point, with at least 100 transfected cells per field).