Figure 12.
FBXO25 prevents aggregation of polyQ-containing proteins. (A) HeLa cells transfected with EGFP-httEx1-103Q were fixed and labeled with anti-FBXO25 antibodies. Images were taken by confocal microscopy, and the labeled proteins are indicated on top of each panel with the respective color. (B) Slot-blot (S.B) filter retardation assays of polyQ aggregates formed in HEK293T cells cotransfected as indicated in B (top). Results shown correspond to two of four sets of independently cotransfected cells. The EGFP-httEx1-74Q protein-containing aggregates were detected with anti-GFP antibodies. (C) Western blotting (W.B) analysis of cell lysates of one set of HEK293T cells cotransfected as indicated in B. The different forms of FBXO25 protein, indicated by arrows, were revealed with ati-FBXO25 antibodies. Input: lysates of control, WT and ΔF cells. (D) Densitometric analysis of the slot-blot membranes prepared with samples of lysates from all four sets of cotransfected cells indicated in B. Results are expressed as percentage of aggregated protein in the control samples. (E) Detection of EGFP-httEx1-74Q mRNAs by RT-PCR from HEK293T cells cotransfected as indicated in E (top). Coexpression of full-length wild-type or mutant version of FBXO25 had no effect on expression of EGFP-httEx1-74Q mRNA. (F) S.B filter retardation assays of polyQ aggregates formed in HEK293T cells that were transfected with either Cul1WT or Cul1DN and EGFP-httEx1-74Q, FBXO25WT, Skp1, and Roc1. Results shown correspond to two of four sets of independently cotransfected cells. (G) W.B analysis of cell lysates of one set of HEK293T cells cotransfected as indicated in F. The different forms of FLAG-Cul1 protein, indicated by arrows, were revealed with anti-FLAG antibodies. Input, lysates of Cul1WT and Cul1DN cells. (H) Densitometric analysis of the slot-blot membranes prepared with samples of lysates from all four sets of cotransfected cells indicated in F. Results are expressed as percentage of aggregated protein in the Cul1DN.