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Fig. 4
Testing for possible alternative translational initiation downstream from the mutation site.
(A) Construction of the PAK4-GFP fusions. The 52-UTR and open reading frame of pak4 were cloned upstream of an eGFP cassette. The TALEN mutation site is indicated by the red arrow. (B) Expression of the fusions in zebrafish embryos. PAK4-eGFP RNAs were synthesized by in vitro transcription and injected into one-cell stage embryos. Green fluorescence was monitored at 24 hpf. Only the wild type construct exhibited fluorescence. (C) Western blot analysis of PAK4-eGFP RNA-injected embryos. Embryos at 24 hpf were dechorionated and used for total protein extraction and Western analysis with a GFP-specific antibody. eGFP RNA was injected into embryos as a positive control. UI, uninjected wild type EK. wt, wild type PAK4-eGFP. Δ2, PAK4Δ2-eGFP. Δ4, PAK4Δ4-eGFP. Ponceau Red staining of the Western blot membrane is shown as a loading control.