Transcription Factor Sequestration by Polyglutamine Proteins - Protein Misfolding and Cellular Stress in Disease and Aging - page 215

Fig. 3. Example data of immunofluorescence microscopy showing co-localization of
transcriptional factor with mutant Htt inclusion in Neuro2a cells. Neuro2a cells were
transfected with expression vector for NF-YA or LacZ (control) tagged with V5 together
with expression vector for Nhtt18Q-EGFP-NLS or Nhtt150Q-EGFP-NLS. After 24 h, cells
were fixed and stained with monoclonal anti-V5 antibody. Nhtt proteins were detected
as GFP fluorescence. Nuclei were detected with DAPI. Note that Nhtt150Q-EGFP-NLS
but not Nhtt18Q-EGFP-NLS, formed nuclear inclusions and NF-YA-V5 preferentially
localized to Nhtt150Q-EGFP-NLS inclusions in Neuro2a cells.

2. Wash the cells with PBS, and permeabilize the cells with 0.5 ml
of 0.1% TritonX-100/PBS for 10 min at room temperature.
3. Wash the cells with PBS, and incubate the cells with 0.5 ml of
10% goat serum/TBST (see Note 8).
4. After washing with TBST, incubate the cells with anti-V5
antibody diluted with 0.1% BSA/TBST for 1 h at 37°C.
5. Wash the cells with TBST three times for 10 min each.
6. Incubate the cells with anti-mouse IgG conjugated with Alexa
546 diluted with 0.1% BSA/TBST for 1 h at 37°C.
7. Wash the cells with TBST three times for 10 min each.
8. Incubate the cells with 10 mM DAPI/TBST for 10 min at
room temperature.
9. After brief washing with TBST, cover the cells by a coverslip
with VECTASHIELD Mounting Medium.
10. Analyze the cells with fluorescence microscope or confocal
laser microscope (see Fig. 3 ).

Even though you could find the interaction of transcriptional
factor with polyglutamine aggregates in transfected cultured cells,
you have to confirm the interaction in vivo to show the patho-
logical significance of this interaction. Currently, mouse models
for HD and some other polyglutamine diseases have been estab-
lished and used as in vivo models. For the HD model mouse,
R6/2 mouse, a heterozygous transgenic mouse carrying htt exon
1 with 150 CAG repeats, is widely used and can be obtained from

3.6. Analysis
of Sequestration
of Transcriptional
Factors
by Polyglutamine
Aggregates in HD
Model Mouse Brain