Fig. S3

Pura in zebrafish embryos. (a) RNA pull-down from mouse brain lysate. Western blot analysis of Pur-alpha, hnRNPA1 and hnRNPH1, showing the binding affinity to different sense and antisense repeat RNAs, n = 2 biological replicates (Input = brain lysate, MW = molecular weight marker). (b,c) Effect of Pur-alpha (0.573μM) on ~70AS (0.844 μM) axonal toxicity (n = 6 experiments). (b) Data represent mean +/- 95% CI, one-way ANOVA, F(2, 262) = 10.52, *p<0.05, ****p<0.0001. (c) Data represent mean +/- 95% CI, logistic regression (z-values compared to GFP: 2.782, 2.308; 70AS + GFP vs 70AS + Pur-alpha: z-value = 1.043, p = 0.297), *p<0.05, **p<0.01. (d,e) Effect of Pur-alpha (0.573μM) on 108RO (0.844 μM) axonal toxicity (n = 8 experiments). (d) Data represent mean +/- 95% CI, one-way ANOVA, F(2, 357) = 10.89, **p<0.01, ****p<0.0001. (e) Data represent mean +/- 95% CI, logistic regression (z-values compared to GFP: 2.521, 0.712; 108RO + GFP vs 108RO + Pur-alpha: z-value = -2.065, p = 0.0389), *p<0.05. (f) RNA pull-down from mouse brain lysate. Western blot analysis of Pur-alpha, showing the binding affinity to different DPR coding codon-optimized mRNAs, n = 2 biological replicates. (Input = brain lysate, MW = molecular weight marker, Negative Control = non-biotinylated ~70S repeat RNA). Hsp60 was used as negative control for the RNA pull-down. (g, h, i, j) Whole mount immunohistochemistry (pura antibody, Hoechst) of 30 hpf zebrafish embryos injected with 3S (h), ~70AS (i) or ~90S (j) and non-injected embryos (g). Arrowheads indicate sites of colocalization of pura with repeat RNA.