Tcf20 interacts with mutant Nhtt aggregates through its N-terminal region in neuro2a cells.

<p>(A) Primary sequences of mouse Tcf20. Glutamine (Q) residues were marked by red. Note that several polyQ stretches are observed in its N-terminal region. (B, C) Neuro2a cells were transfected with expression vectors for Nhtt150Q-EGFP-NLS (B) or Nhtt150Q-EGFP (C) together with those for indicated V5-tagged proteins. After one day, the cells were fixed and stained with anti-V5 antibody and Alexa 546-conjugated anti-mouse IgG. Nuclei were stained with DAPI. (B) Tcf20 co-accumulated with Nhtt150Q-EGFP-NLS aggregate in nucleus whereas it diffusely located in the nucleus without the aggregate. (C) Tcf20 or its N-terminal fragments accumulated with Nhtt150Q-EGFP aggregates in cytoplasm whereas Rab2b or LacZ did not. (D) Neuro2a cells were co-transfected with Nhtt150Q-EGFP expression vectors together with those for indicated V5-tagged proteins. After one day, the cells were subjected to Western blot analysis using anti-V5 and anti-GFP antibodies. Bands for expressed proteins are indicated by arrowheads. Note that gel top bands (indicated by arrows) were observed for Tcf20 (1∼400 or 1∼500)-V5 and NF-YA-V5 but not for LacZ-V5 by co-expression of Nhtt150Q-EGFP, suggesting their co-insolubilization with mutant Nhtt in neuro2a cells. (E) Nhtt150Q-EGFP cells were transfected with expression vectors for Tcf20 (full, 1∼400 or 1∼500)-V5 together with pRFP-C1 vector. After one day, cells were treated with ponasterone A for 24 hr and the cells with aggregates in trasnfected (RFP-positive) cells were quantified by ArrayScan reader. Ratios to control (empty vector) are shown. Values are means ± SD of four well data (**<i>P</i><0.01). Scale bars are 10 μm (B, C).