Transcription Factor Sequestration by Polyglutamine Proteins - Protein Misfolding and Cellular Stress in Disease and Aging - page 220

2. If the expression level of the transcriptional factor seems to be
much higher or lower than that of mutant Htt in transfected
cells, it may be difficult to observe the interaction between
these. In such a case, change the ratio of plasmid DNA
for transcriptional factor to that for Nhtt150Q-EGFP-NLS.
If this does not solve the problem, try other experimental
conditions, such as the use of a different promoter for expres-
sion of transcriptional factor, or use of different type of cell
for transfection, etc.
3. In the case of Nhtt150Q-EGFP-NLS, distinct nuclear inclu-
sions are observed at 24 h after transfection ( 11 ). If you
expressed other polyglutamine protein and could not observe
inclusions at this time point, culture the cells one more day
after changing the medium to new one. Alternatively, you can
add dibutyryl cyclic AMP to medium to a final concentration
of 5 mM in step 7. Because dibutyryl cyclic AMP suppresses
cell proliferation to differentiate Neuro2a cells, the formation
of polyglutamine aggregates is usually enhanced.
4. This overnight incubation seems to be critical for observation
of aggregated proteins by Western blot analysis and filter trap
assay. This may be due to low efficiency of interaction of anti-
body with the aggregates.
5. If the lysates are too sticky to go through the membrane, use
lower amount of lysates. Brief sonication after step 2 may also
avoid this problem.
6. This step seems to be necessary to fix the aggregates on the
membrane well.
7. Because GFP is light sensitive, shield the slide chamber
from light.
8. Alternatively, 5% skim milk/TBST can be used.
9. You can use mouse monoclonal antibody against htt
(MAB5374: Chemicon) instead of anti-ubiquitin if the anti-
body against transcriptional factor is not derived from mouse.
10. You can store the sections in the freezer, but the frozen sec-
tions should be dried thoroughly before use. Without doing
this, sections will be easily detached from the slide glass.
11. During this step, the transcriptional factors will be dissolved
into the buffer.
12. If the protein concentration of the lysates is lower than
20 mg/5 ml, lower concentration (e.g. 10 mg/5 ml) is also
possible. However, use of lysates with low concentration will
reduce the signal of shifted band in EMSA.
13. If the transcriptional factor is sequestrated by mutant Htt
aggregates, reduction of its protein level in R6/2 tissue lysates
would be observed.