Transcription Factor Sequestration by Polyglutamine Proteins - Protein Misfolding and Cellular Stress in Disease and Aging - page 211

9. Poly(dI-dC).
10. Bovine serum albumin.
11. Polyacrylamide gel apparatus.
12. 4% native polyacrylamide gel (2.7 ml of 30% acrylamide
(29:1), 1 ml of 5× TBE, 0.2 ml of 10% ammonium sulfate
(APS), 20 ml of N,N,N¢,N¢-Tetramethylethylenediamine
(TEMED), 16.1 ml of H 2 O for 20 ml gel).
13. 5× TBE (53.89 g of Tris, 27.5 g of Boric acid, 3.72 g of
EDTA·2Na (2H 2 O) and H 2 O up to 1 L).
14. 0.25× TBE running buffer.
15. Loading dye (0.4% bromophenol blue, 0.2% xylene cyanol,
50% glycerol).
16. X-ray film or Imaging Plate (Fijifilm).

3. Methods

The easiest way to analyze the interaction between polyglutamine
aggregates and transcriptional factors is the use of transfected cul-
tured cells. Here, we introduce three methods to examine the
interaction of transcriptional factors with mutant Htt aggregates
in cultured cells. For mutant Htt expression construct, pcDNA3.1
expression vector encoding Nhtt150Q-EGFP-NLS is used in the
following methods ( 12 ). pcDNA3.1 expression vector encoding
Nhtt18Q-EGFP-NLS is used as a control. By subcloning the
cDNA for the transcriptional factor of your interest into
pcDNA3.1-V5-His, you can detect the transcriptional factor by
anti-V5 antibody. Alternatively, other tags such as Myc, HA are
also available. In the following experiments, V5 tagged transcrip-
tional factor is used as an example.

3.1. Analysis
of Interaction
of Transcriptional
Factors with
Polyglutamine
Aggregates in
Cultured Cells

3.2. Neuro2a Cell
Transfection

We often use Neuro2a cells, a mouse neuroblastoma cell line, for
transfection experiments because they are easy to handle, grow
very fast and are very efficiently transfected.

1. Seed 8 × 10 4 of Neuro2a cells on 24 well plate with 0.5 ml of
10% FBS/DMEM and incubate the cells overnight at 37°C
in a CO 2 incubator (see Note 1).
2. Mix 0.4 mg of pcDNA3.1-Nhtt150Q-EGFP-NLS or
Nhtt18Q-EGFP-NLS and 0.4 mg of plasmid encoding
transcriptional factor with 50 ml of Opti-MEM in a tube (see
Note 2). In another tube, mix 2 ml of lipofectamine 2000
reagent with 50 ml of Opti-MEM.
3. Incubate these mixtures at room temperature for 5 min.