Overexpression of mutant ATXN2 in human SH-SY5Y cells recapitulates down-regulation of <i>in vivo</i> steady-state levels of Rgs8 in BAC-Q72 mice.

<p>Cells were transfected with plasmids encoding Flag-tagged cDNAs of human <i>ATXN2</i> containing Q22 or Q58 or Q108 repeats. Forty-eight hrs post-transfection, cells were selected with hygromycin (40 µg/ml) for 5–7 days and hygromycin resistant cells were harvested as two aliquots. <b>(A)</b> Protein extracts were prepared from one aliquot and subjected to Western blot analyses to measure steady-state levels of RGS8. The blots were re-probed for β-Actin as an internal loading control. <b>(B)</b> Quantitative RT-PCR analyses of synthesized cDNAs from the other aliquot demonstrate moderate reduction of <i>RGS8</i> mRNA in cells expressing Flag-ATXN2-Q108. The data are means ± SD, *p<0.05. <b>(C)</b> Mutant ATXN2 specifically induces decrease of RGS8 expression. MYC-tagged <i>RGS8</i> cDNA including 5’ and 3’ UTRs was cloned under the transcriptional control of the CMV promoter and transfected into short-term hygromycin selected SH-SY5Y cell lines expressing Flag-tagged ATXN2-Q22, -Q58 or -Q108. Forty-eight hrs post-transfection, levels of exogenous RGS8 are significantly decreased in cells expressing ATXN2-Q58 or -Q108 compared with cells expressing wild-type ATXN2-Q22. To control for equal transfection, we monitored levels of GFP, which was expressed as an independent cassette in the plasmid. Blots were re-probed for β-actin as an internal loading control. The blot represents one of three independent experiments.