Figure 5
Interaction of ITCH with expanded polyglutamine proteins.
(A–B) Fluorescence micrographs of Cos-7 cells showing the expression and aggregation of expanded-polyglutamine ataxin-3(Q84) (top) and HDQ74 (bottom) proteins (A). Lysates were collected from the same sets of cells and subjected to immunoprecipitation (IP) with the anti-GFP antibody and the blots were sequentially developed using anti-GFP and anti-ubiquitin antibodies. (C) Cells expressing normal EGFP-HDQ23 and expanded-repeat EGFP-HDQ74 huntingtin were used for coimmunoprecipitation analysis. The cell lysates were pulled down by the anti-ITCH antibody and the blots were probed with anti-GFP and anti-ITCH antibodies. (D) Transient transfection was used to overexpress normal ataxin-3 (ataxin-3(Q28)) and ataxin-3 with expanded polyglutamine repeats (ataxin-3(Q84)). After 48 h of transfection, the cell lysates were prepared and processed for immunoprecipitation (IP) with the anti-ITCH antibody and sequentially, the blots were developed using anti-GFP and anti-ITCH antibodies. (E–F) Lysates of cells expressing normal and expanded-polyglutamine versions of ataxin-3 (E) and huntingtin (F) were pulled down by beads only (control) and immunoblotting was performed with an anti-GFP antibody.
