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. 2017 Sep 13;37(37):9000–9012. doi: 10.1523/JNEUROSCI.0118-17.2017

Figure 4.

Figure 4.

mHtt is targeted preferentially to LE/Lys compared with wtHtt. A, Enrichment of mHtt over wtHtt in the light membrane fraction. Neuro2A-mHtt and Neuro2A-wtHtt cells were homogenized and subjected to floatation in sucrose density gradients. Then, 2 μg of light membrane (LM) (left) and cytosolic (cyt) proteins (center) were analyzed by Western blotting using anti-Htt antibody. Lamp1 and tubulin were used as loading controls for the light membranes and cytosols, respectively. Right, Ratio of light membrane and cytosolic Htt expressed as a percentage of mHtt. n = 3, p = 0.0222. B, Increased targeting of mHtt over wtHtt to Lamp1-positive vesicles. Left, Neuro2A-mHtt (top) or Neuro2A-wtHtt (bottom) cells were pre-permeabilized, fixed, immunolabeled with anti-Htt and anti-Lamp1 antibodies, and analyzed by confocal microscopy. DAPI was used to visualize nuclei. Scale bar, 10 μm. Top right, Htt integrated density per cell expressed as a percentage of mHtt. n = 4, p = 0.0001. Bottom right, Htt/Lamp1 colocalization. n = 3, p = 0.0036. Error bars indicate SD. *p < 0.05; **p < 0.01; ***p < 0.001.