FIGURE 8.

p62 is recruited to Ataxin1Q84 nuclear protein inclusions and facilitated the recruitment of proteasomes to these structures. A, quantification is shown of colocalization between nuclear GFP-Ataxin1Q84 inclusions and endogenous p62 in HeLa cells 24 and 48 h after transfection. HeLa cells were transiently transfected with the expression vector for GFP-Ataxin1Q84. 24 and 48 h after transfection cells were fixed, stained with antibodies against p62, and scored for colocalization between p62 and GFP-Ataxin1Q84. Each bar represents the mean ± S.D. of 3 independent experiments with more than 100 cells counted per experiment. B and C, confocal images are shown of HeLa cells transiently transfected with GFP-Ataxin1Q84, stained with antibodies against p62, PML, proteasome 20S core subunits, and polyubiquitin (polyUb) (FK1) 48 h after transfection. Scale bars equal 10 μm D, wild type (WT) or p62^−/− MEFs were transiently transfected with the expression vector for GFP-Ataxin1Q84. 24 and 48 h after transfection, cells were fixed, stained with antibodies against the proteasome 20 S core subunits, and scored for colocalization between GFP-Ataxin1Q84 nuclear inclusions and proteasome staining by fluorescent microscopy. Each bar represents the mean ± S.D. of 3 independent experiments with more than 100 cells counted per experiment. E, HEK293 cells stably transfected with GFP-Ataxin1Q84 construct under the control of tet repressor-regulated CMV promoter were transfected with control (Ctrl.) siRNA or siRNA against p62, and the expression of GFP-Ataxin1Q84 was induced by tetracycline for 24 h. At 0, 12, 24, 48, and 72 h after removal of tetracycline, cells were lysed and subjected to SDS-PAGE and immunoblotting (WB) with the indicated antibodies. F, shown is quantification of the rate of GFP-Ataxin1Q84 degradation from E. The levels of GFP-Ataxin1Q84 were normalized to actin. Each bar represents the mean ± S.D. of three independent experiments. No ind., no induction.