Figure 3

Recruitment of ITCH with cytoplasmic expanded-polyglutamine huntingtin inclusion bodies.

(A–B) Cos-7 cells were plated onto two-well chambers and transiently transfected with EGFP-HDQ23 (A) and EGFP-HDQ74 (B) constructs. Forty-eight hours after transfection, the cells were subjected to immunofluorescence staining with the anti-ITCH antibody. (C–D) Colocalization of ubiquitin (red) with cytoplasmic inclusions of misfolded polyglutamine EGFP-HDQ74 (D) proteins compared to the EGFP-HDQ23 (C) with normal repeats is shown. (E–F) Similar sets of cells were subjected to LC3 (red) staining for normal EGFP-HDQ23 (E) and EGFP-HDQ74 (F) with expanded repeats. (G–H) In several experiments, the cells were processed for immunolocalization of cytoplasmic huntingtin aggregates with p62 (red) in normal (G) and expanded- (H) polyglutamine-expressing cells. (I–J) The cells that expressed EGFP-HDQ23 (I) and EGFP-HDQ74 (J) constructs were subjected to immunofluorescence staining with an antibody targeting the 20S proteasome. A rhodamine-conjugated secondary antibody (red) was used to label ITCH, ubiquitin, LC3, p62 and 20S proteins. The arrows indicate the redistribution of ITCH with cytoplasmic polyglutamine-expanded huntingtin aggregates positive for p62, ubiquitin, LC3 and 20S. The overlay images also include 4′,6-diamidino-2-phenylindole (DAPI) staining of the cell nuclei. Scale bar, 20 μm.