Figure 4
Recruitment of ITCH by cytoplasmic ataxin-3 aggregates.
(A–B) Cells were transiently transfected with ataxin-3-GFP fusion with normal (A) (ataxin-3(Q28)) or expanded (B) polyglutamine repeats (ataxin-3(Q84)). Forty-eight hours after transfection, the cells were processed for immunofluorescence staining with an anti-ITCH antibody (red). (C–D) Inclusion bodies (IBs) containing ataxin-3 with expanded polyglutamine repeats were immunopositive for ubiquitin. Micrographs of Cos-7 cells were transiently transfected with normal (C) ataxin-3(Q28) and expanded- (D) polyglutamine ataxin-3(Q84) plasmids and the cells were probed with an anti-ubiquitin antibody (red). (E–F) Analysis of expanded-polyglutamine ataxin-3-positive inclusion bodies (IBs) with LC3. The same sets of cells were subjected to immunofluorescence staining with an anti-LC3 (red) antibody in normal (E) and expanded- (F) polyglutamine repeat-expressing cells. (G–H) Recruitment of p62 by ataxin-3 with expanded polyglutamine repeats. Micrographs represent cells transiently transfected with normal (G) ataxin-3(Q28) and expanded- (H) polyglutamine ataxin-3(Q84) plasmids. After 48 h, the cells were subjected to immunofluorescence staining with an anti-p62 (red) antibody. (I–J) Colocalization of 20S proteasome with ataxin-3 containing expanded polyglutamine repeats. Cells that expressed normal (I) and expanded-polyglutamine (J) ataxin-3 were used for immunofluorescence staining with an anti-20S proteasome antibody. A rhodamine-conjugated secondary antibody (red) was used to label ITCH, ubiquitin, LC3, p62 and 20S proteins. The arrows indicate colocalization of ITCH with cytoplasmic inclusion bodies (IBs) containing ataxin-3 with expanded repeats that were also positive for ubiquitin, LC3, p62 and 20S. The overlay images also include 4′,6-diamidino-2-phenylindole (DAPI) staining of the cell nuclei. Scale bar, 20 μm.
