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Fig. 1

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ZDB-IMAGE-130118-19
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Figures for Kaiser et al., 2012
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Fig. 1 Knockdown of Appa, Appb, or Prp1 results in impaired development and death of head region. A–L.

Morpholino (MO) was delivered to disrupt translation of endogenous amyloid β precursor protein (APP) and prion protein (PrP) paralogs in zebrafish: appa, appb, or prp1 (top-bottom rows, respectively). Standard control MO at levels equivalent to our effective dose fail to induce any CNS cell death or disruptions in morphology of the fish (left column). Low doses of appa, appb, or prp1 MOs (0.5, 1.0, 0.5 ng respectively) were empirically determined to be sub-effective (Fig. S1,S2), leading to mild changes, but no death of CNS tissues (2nd column). Effective doses (1.0, 2.5, 1.0 ng, respectively) lead to severe alterations in CNS morphology (*) and death of CNS tissues (3rd column). Specificity of the MOs was demonstrated by rescuing the injection of an effective dose of appa, appb, or prp1 MO by co-injection of the cognate mRNA (200 pg, 200 pg or 100 pg, respectively; Right column). These data are quantified in Figs. 2F, S1 & S2. M. Western blots of zebrafish lysates reveal efficacy of our MO knockdown reagents (see also Fig. S3). The appa and appb splice blocking (SB) MOs used above (A–H) significantly decreases detection of protein species by the antibody 22C11 (top row). Bottom row is a β-actin loading control. An additional, independent MO that acts as a translation block of appa (appa TB) confirms this protein knockdown and produces similar phenotypes (Fig. S1). The prp1 MO reagents used here were previously shown to be effective in knocking down protein [27]. N. Quantification of western blots from three biological replicates (three independent injection trials on three separate days) demonstrate a significant decrease (*p<0.05, **p<0.01) of the APP immunoreactivity compared to β-actin with all three MO reagents at their effective doses.

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