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. 2012 Aug 21;21(23):5048–5065. doi: 10.1093/hmg/dds349

Figure 2.

Figure 2.

Effect of CAG length on ATXN2 expression. (A) Luciferase assays using ATXN2-luc constructs with increasing CAG repeat lengths revealed highest expression for CAG22 and CAG57 and lower expression for longer CAG lengths. All constructs were based on pGL2-5A3. Identical results were obtained when the experiment was repeated using constructs modified to include TK-Renilla cassettes on the same plasmids. (B) Western blot analysis of the constructs in (A) revealed a similar pattern of protein expression to that determined by luciferase assays in (A). Note that lane 2 was purposefully underloaded. (C) qPCR analysis using constructs shown in (A) that were modified to include TK-Renilla cassettes on the same plasmids, showed no changes in the abundances of the ATXN2-luc transcript depending upon the CAG length. (D) Constructs expressing full-length ATXN2 with repeat lengths CAG22, CAG58 and CAG108 including 4 kb ATXN2 upstream sequence, analyzed by western blotting with anti-FLAG antibody. Like in (A) and (B), the longest repeat (CAG108) reduced ATXN2 expression. The asterisk indicates a significant difference for CAG108 versus either CAG22 or CAG58 (P < 0.05, Bonferroni post-test, n = 7 transfections). Expression was relative to GFP encoded on the same plasmid as indicated. Actin is also shown. HEK293 cells (A–C) and SH-SY5Y cells (D).