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Fig. S1
Testing of Anti-P54 serum and P54a and P54b morpholinos. Testing of Anti-P54 serum and P54a and P54b morpholinos. (A) Western blot analysis using protein extracts from 24 hpf WT zebrafish embryos (12 embryos per lane): the rabbit pre-immune serum (dilutions 1/500 – 1/3000) produces no specific signal, whereas the anti-P54 serum (dilutions 1/500 –1/3000) showed a band at approximately 54 kDa, which is the predicted molecular weight of both P54 RNA helicases from zebrafish. (B) Fragment of a protein alignment between P54a and P54b with the sequence used to synthesize the peptide against anti-P54 serum was generated. (C and D) Wholemount immunostaining of WT embryos at 24 hpf using 1/2000 dilution of anti-P54 serum or rabbit pre-immune serum. (E) Protein extracts from 24 hpf embryos micro-injected with MO-P54ab or MO-ct were tested by Western blotting with the anti-P54 serum, and the MO-P54ab embryos show a reduction in the signal. As a loading control, an anti-tubulin antibody was used. (F) Relative density measured in anti-P54 protein bands in both MO-P54ab and MO-Ct total protein samples, normalized using the anti-Tubulin loading controls. (G – I) Immunostaining of MO-ct or MO-P54ab 24 hpf morphants showed the loss of the P54 signal in the MO-P54ab-treated embryos, but this signal could be recovered by rescuing P54a expression by co-injecting a mix of MO-P54ab and mRNA-P54a. Nuclei were counterstained with DAPI. The percentage of embryos showing the same pattern shown in the figure is indicated in each panel, with the total number of embryos tested indicated in parentheses. Control morpholino (MO-ct), mix of p54a and p54b morpholinos (MO-P54ab), “in vitro” synthesized mRNA from p54a (mRNA-P54a).