Transcription Factor Sequestration by Polyglutamine Proteins - Protein Misfolding and Cellular Stress in Disease and Aging - page 214

Fig. 2. Example data of filter trap assay showing interaction of transcriptional factor with
SDS-insoluble aggregates in Neuro2a cells. Neuro2a cells were transfected with expres-
sion vector for NF-YA tagged with V5 together with expression vector for Nhtt18Q-EGFP-
NLS or Nhtt150Q-EGFP-NLS. After 24 h, cells were lyzed and subjected to filter trap
assay and stained with anti-GFP or anti-V5 antibody. Note that spots were detected by
anti-GFP and anti-V5 staining in the cells expressing Nhtt150Q-EGFP-NLS but not ones
expressing Nhtt18Q-EGFP-NLS.

9. Transfer membrane into blocking buffer and incubate it for
30 min at room temperature.
10. After brief washing with TBST, incubate the membrane with
primary antibody against GFP or V5 diluted in 3% goat
serum/TBST overnight at 4°C (see Note 4).
11. Wash the membrane with TBST three times for 10 min each.
12. Incubate the membrane with HRP conjugated secondary
antibody diluted in 3% goat serum/TBST for 1 h at room
temperature.
13. Wash the membrane with TBST three times for 10 min each.
14. Detect the protein using chemiluminescent reagent as recom-
mended by the manufacturer (see Fig. 2 ).

3.5. Immunofluor-
escence Microscopy

The last method to analyze the interaction of transcriptional fac-
tor with mutant Htt aggregates in transfected cells is immuno-
fluorescence microscopy. If the transcriptional factor is
incorporated into mutant Htt aggregates, you can observe the
co-localization of transcriptional factor with mutant Htt-EGFP
inclusions. This method may be also useful for analysis of a weak
and/or SDS-sensitive interaction between transcriptional factor
and mutant Htt aggregates which may be hardly detected by
Western blot analysis or filter trap assay described above. If the
transcriptional factor is efficiently sequestered by mutant Htt
aggregates, reduction of diffuse staining will be observed (see
Fig. 3 ). In this experiment, you can use 4 well chamber slide
instead of 24 well plastic plate for analysis with fluorescence
microscope or confocal laser microscope.

1. Wash the transfected cells grown on a four well chamber slide
with PBS, and fix the cells with 0.5 ml of 4% PFA/PBS for
10 min at room temperature (see Note 7).