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. 2014 Oct 24;24(5):1363–1373. doi: 10.1093/hmg/ddu545

Figure 5.

Figure 5.

In vivo effects of hGle1-S70X and hGle1-IVS14-2A>C mutants. (A) Graph showing the extent of head cell necrosis at 32 h post-fertilization (scored as severe, mild or WT-like) in zebrafish embryos that were injected with two non-overlapping zGle1 translation-blocking morpholinos (zGle1 MOs) followed by either buffer alone or 150 pg of FLAG-tagged human GLE1B-WT, GLE1-c.209C>A or GLE1-1965-2A>C mRNA at the one-cell stage. Values graphed as ‘mean ± SEM’, n = number of embryos analyzed in each condition. (B) Confocal images of live 2-day-old control zebrafish and zGle1 morphants (zGle1 MOs) that were injected with buffer alone (zGle1 MOs) or with 150 pg of FLAG-tagged human GLE1B-WT, GLE1-c.209C>A or GLE1-1965-2A>C mRNA at the one-cell stage. Motoneurons were labeled by the mnx1:TagRFP-T transgene, and images are lateral views of the trunk spinal cord with the dorsal side to the top and the anterior side to the left. Scale bars: 50 µm. (C) Western blot analysis of hGle1 expression in zGle1 morphants injected with buffer alone (no RNA) or 150 pg of FLAG-tagged GLE1B-WT, GLE1-c.1965-2A>C or GLE1-c.209C>A mRNA. Blots probed with rabbit anti-hGle1 antibody (which does not recognize the zebrafish Gle1 protein). Actin was used as a loading control. (D) Graph showing rate of degradation of hGle1B-WT versus hGle1-IVS14-2A>C and hGle1-FinMajor Flag-tagged proteins in HeLa cells in the presence of the translation inhibitor cycloheximide (*P ≤ 0.05, ***P ≤ 0.001).