Transcription Factor Sequestration by Polyglutamine Proteins - Protein Misfolding and Cellular Stress in Disease and Aging - page 212

4. Mix these two and incubate at room temperature for
20 min.
5. Add the mixed solution (100 ml) to cells.
6. Incubate the cells for 4-6 h at 37°C in a CO 2 incubator.
7. Change the medium to 0.5 ml of DMEM supplemented with
10% FBS and penicillin-streptomycin.
8. Incubate the cells for 20-24 h at 37°C in a CO 2 incubator
(see Note 3).

3.3. Western Blot
Analysis

Nhtt150Q-EGFP-NLS but not Nhtt18Q-EGFP-NLS forms
SDS-insoluble aggregates in the transfected Neuro2a cells ( 11,
12 ). Because such insoluble materials do not migrate into the gel
during SDS-PAGE, they can be detected in the gel top by Western
blot analysis using antibody against the GFP-portion of the fusion
protein. If the transcriptional factor is also incorporated into the
aggregates and becomes SDS-insoluble, it can be detected in the
gel top similar to mutant Htt aggregates.

1. Wash the transfected cells with PBS and harvest the cells with
150 ml of 2× SDS sample buffer.
2. Heat the cell lysates at 100°C for 5 min.
3. Sonicate the lysates with a microsonicator for about 10 s.
4. Resolve the proteins through SDS-PAGE and transfer them
onto PVDF membrane using standard electrophoresis and
blotting procedures.
5. Incubate the membrane in blocking buffer (5% skim milk/
TBST) for 30 min at room temperature.
6. 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).
7. Wash the membrane with TBST three times for 10 min
each.
8. Incubate the membrane with HRP conjugated secondary
antibody diluted in 3% goat serum/TBST for 1 h at room
temperature.
9. Wash the membrane with TBST three times for 10 min each.
10. Detect the protein by chemiluminescent reagent as recom-
mended by the manufacturer (see Fig. 1 ).

3.4. Filter Trap Assay

Filter trap assay is another way to identify the SDS-insoluble
aggregates ( 13, 14 ). In this assay, cellulose acetate membrane with
0.2 mm pore size is used. If the cell lysates expressing Nhtt150Q-
EGFP-NLS are loaded, only aggregated proteins are trapped in
the membrane and can be detected with anti-GFP. Incorporated
transcriptional factor can be detected by anti-V5. This assay is often