FIGURE 3.
Hydrodynamic assessment of Httex1-Emerald monomers in Neuro2a lysates by sedimentation velocity analysis. A, Western blot shows the expression levels over 2 days. Only Httex146Q-Emerald forms SDS-insoluble material over 2 days, indicative of aggregates. B, confocal microscopy shows Httex125Q-Emerald to remain distributed evenly through the cytosol of Neuro2a cells after 2 days. In contrast, the Httex146Q-Emerald forms punctate IBs in many cells. C, high speed sedimentation velocity analysis (50,000 rpm) of Httex1-Emerald in the lysate (0.5 mg/ml total protein) after 2 days of expression. The first scan is shown in red, with subsequent scans in incrementing grayscale. The first scan shows a fast pile up of material at the bottom for Httex146Q-Emerald for a fraction of the material. The lower panels show residuals to fits to a c(s) distribution. D, the sedimenting boundaries for Httex1-Emerald dictate a highly monodisperse population (elongated monomers) when assessed by c(s) analysis (upper panel) or van Holde-Weischet analysis (lower panel). Zoomed in rescaling of the c(s) distributions shows a minor fraction of material to sediment at 3–8 S (inset of upper panel).