Figure 6
Redistribution and colocalization of the ITCH mutant with misfolded proteins and expanded polyglutamine proteins.
(A–B) The inactive ITCH mutant (Myc-ITCH-C822S) was co-expressed with GFP-wtCAT (A) and GFP-Δ9CAT (B) constructs. The cells were processed for immunofluorescence staining with an anti-Myc antibody and nuclear staining was detected using DAPI. Arrows indicate the colocalization of the ITCH-C822S mutant form with cytoplasmic misfolded GFP-Δ9CAT-protein aggregates. (C–D) As depicted in the figure, normal (C) (ataxin-3(Q28)) and expanded- (D) polyglutamine (ataxin-3(Q84)) ataxin-3 constructs were transiently cotransfected in two-well chamber slides with the ITCH mutant construct. After 48 h of transfection, the cells were subjected to immunofluorescence staining with an anti-Myc antibody and the nuclei were stained with DAPI. Recruitment of the ITCH mutant form by cytoplasmic inclusion bodies containing ataxin-3 with expanded polyglutamine repeats is indicated by the arrows. (E–F) The expression constructs encoding EGFP-HDQ23 (E) with normal polyglutamine repeats and EGFP-HDQ74 (F) with expanded repeats were expressed with Myc-ITCH-C822S constructs and the transfected cells were processed for immunofluorescence with an anti-Myc antibody. The arrows indicate the colocalization of the ITCH mutant form with huntingtin HDQ74 cytoplasmic aggregates. A rhodamine-conjugated secondary antibody (red) was used to label Myc-ITCH-C822S. Scale bar, 20 μm.
