The construction of the Igfbp5a expression plasmid has been reported (3). This plasmid was used as a template to generate Igfbp5a NLS and LBD mutants. For the NLS mutant, the 236KGRK239 motif in Igfbp5a C-domain was changed to LDGQ, the corresponding sequence of zebrafish Igfbp1a (58) by site-directed mutagenesis (table S1). For the LBD mutant, the sequence 86KPLLL92 in Igfbp5a was mutated to NQQQQ (59). Human IGFBP5 G223R and IGFBP5 W242* were generated by site-direct mutagenesis (table S1). The resulting Igfbp5a/IGFBP5 DNAs were cloned into the pIRES2-mCherry using the Eco RI and Bam HI sites. The pIRES2-mCherry plasmid was engineered by replacing the DsRed-Express2 cassette in the pIRES2-DsRed-Express2 plasmid with mCherry using the Bst XI and Not I sites. The Igfbp5a/IGFBP5-IRES2-mCherry-KanR cassette DNAs were amplified by PCR using primers listed in table S1. They were inserted into the igfbp5a BAC construct (4) to replace the igfbp5a sequence from the start codon to the end of the first exon through homologous recombination. The resulting BAC DNA was validated by DNA sequencing using primers shown in table S1. The validated BAC(igfbp5a:Igfbp5a/IGFBP5-mCherry) DNA constructs were injected into igfbp5a−/−;Tg(igfbp5a:GFP) embryos at the one-cell stage. The embryos were raised in the E3 solution until 72 hpf and then subjected to the low-[Ca2+] challenge test by transferring into low-[Ca2+] solution. Cells coexpressing mCherry and GFP were defined as igfbp5a/IGFBP5-expressing NaR cells. They were scored using the scoring system shown in Fig. 5B. A similar cloning strategy was used to generate BAC(igfbp5a:myr-Akt-mCherry) plasmids. Constructs were confirmed by DNA sequencing at the University of Michigan DNA Sequencing Core Facility.

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