A) Schematic illustrates transgene in zebrafish genome and the workflow for analyzing Lamp1 in WT and nus1 morphant embryos. As shown, the transgene encodes a fusion between lamp1 and monomeric Cherry (mCherry); expression is controlled by the heat shock promoter (hsp701). B,C) Western blot analysis and graphic quantitation of Lamp1 over a time course (0.5-10h) following heat shock induction of its expression show while similar amounts of Lamp1-mCherry are made post-heat shock, protein accumulates in nus1 morphants. D,E) Western blot analysis and graphic quantitation of Npc2 abundance in WT and morphants (using either 0.5 or 1.0 μM morpholino) from 1-6dpf. Red bracket indicate Npc2 doublet; both bands included in the quantitation of relative abundance. For all graphs: n=3 experiments, with 25 embryo per sample per experiment. Error=S.E.M, significance calculated by the student’s t-test where *p<0.05, **p<0.01. F) Confocal analyses of filipin stained WT and nus1 morphant (MO) embryos 6dpf show cholesterol accumulation in the hindbrain, spinal cord (sc), and motor axons of morphant embryo tails. Yellow arrowheads highlight motor neuron cell bodies in ventral spinal cord and their associated axonal projections. Boxed region represents a region of interest (ROI) magnified in the right panel. Scale bars = 50 and 25 μM, respectively. G) Graph represents quantitation of the average and maximum pixel intensities from the hindbrains, spinal cords, and axons of 10-15 embryos from n=3 independent experiments. Error=S.E.M, significance calculated by the student’s t-test where ***p<0.001.
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