HeLa cells were transfected with plasmids using Fugene (Roche Diagnostics).
Live-cell microscopy used a pre-warmed live-cell chamber (Bioptechs) with a peristaltic pump to keep fresh medium circulating, and to keep constant temperature.
FRAP (fluorescent recovery after photobleaching) was measured on a Zeiss confocal microscope using wavelength 488nm for GFP and 458 for CFP. Dual FRAP experiments were run using wavelength 458nm. Fluorescent intensities were determined with LSM software (Zeiss, Laser Scanning Microscope, Version 2.5).
The signal intensity of nucleoplasmic, and small and large ataxin1 inclusions from regions of interest (ROI) (bleached areas) were collected over time and analyzed by scoring initial intensity as zero and final intensity at 1 arbitrary units. Logarithic equations were used to normalized intensity at any given time and to form graphs.