Figure 5: UbS2 mutation leads to reduced ataxin-3 protein levels in Drosophila.
From: Ubiquitin-binding site 2 of ataxin-3 prevents its proteasomal degradation by interacting with Rad23
(a) Quantitative reverse transcriptase (qRT)-PCR results from whole flies expressing the noted versions of UAS-ataxin-3 driven by sqh-Gal4. All flies were heterozygous for UAS-ataxin-3 and sqh-Gal4 transgenes. Red arrows: wild-type (WT) and UbS2* lines that have comparable ataxin-3 mRNA levels. Blue arrows: UbS2* lines that have markedly higher ataxin-3 mRNA levels than WT versions. Experiment performed independently in triplicate, utilizing at least five flies per genotype per experiment. Shown are mean ataxin-3 mRNA levels normalized to WT-1. Error bars: s.d. (b) Left: western blots from whole flies based on qRT–PCR results from a. At least five flies were homogenized per genotype. Driver was sqh-Gal4. All flies were heterozygous for UAS-ataxin-3 and sqh-Gal4 transgenes, as in a. Note that for this blot 1.5 times more lysate was loaded for the line that expresses UbS2*-2 to enable visualization of ataxin-3 protein in this line by western blotting without saturating the signal from other lysates. Right: means of ataxin-3 signal quantified from blots on the left and other independent experiments. P-values are from the Student’s t-test (WT-3 and UbS2*-2) and analysis of variance/Tukey (the other lines). Error bars: s.d. N=3 independently conducted experiments. Flies were 1–3 days old.
