FIGURE 5.

The effect of Thr-3 mutation on Httex1p aggregation in vivo. Flies expressing 97Q, 97QT3A, or 97QT3D were assayed for soluble Htt (anti-Htt; S830; top panel) and actin (bottom panel) levels using quantitative immunoblot (A). The Htt signal was quantified and normalized to the actin signal (LI-COR) (B). Insoluble material from Htt-expressing flies was measured by filter retardation assay (C). Equal amounts of fly extract were spotted onto a nitrocellulose membrane, and stained with Ponceau for total protein (top panel) and then probed with anti-Htt (S830) to detect the amount of insoluble Htt in 97Q versus 97QT3A versus 97QT3D (bottom panel). The Htt signal was measured by LI-COR and quantified in D. Eye imaginal discs from 3rd instar larvae were stained for Elav (red, first column) and Htt (S830; green, middle column) (E). Boxes drawn in the merged (last) column, in E, identify the area of the Htt signal that was converted to grayscale (F) and analyzed for total aggregate load using Scion Imaging software as described under “Experimental Procedures” (G). The scale bars equal 50 μm in E and F. wt, wild-type. Error bars are the S.D. from four or more imaginal discs for each genotype (E and F) and three independent dot blots (C and D). *, p ≤ 0.05; **, p ≤ 0.01.