He alignment is available upon request. Phylogenetic trees were constructed by the neighbor joining (NJ) method using the PHYLIP package [28] with the substitution model and represented with the FigTree software (http://tree.bio.ed.ac.uk/software/figtree/). Tree topology was evaluated by bootstrap resampling with 1000 replicates.the DpnI-resistant chimeric DNA was recovered by transformation into competent bacteria. The chimeric gene was named ssat1a248b, as it contains nucleotides 1?48 of ssat1a and the remainder of ssat1b. Chimeric genes ssat1a332b, ssat1a374b, ssat1a453b, ssat1b248a, ssat1b332a, ssat1b389a, and ssat1b467a were also prepared in this manner (Fig. 2B). The chimeric gene ssat1aba was prepared by using nucleotides 389?13 of ssat1a as the megaprimer and a plasmid containing ssat1a332b as template. Therefore, ssat1aba and ssat1bab contain most of the ssat1a and ssat1b sequences, but their 332?89 nucleotide regions are from ssat1b and ssat1a.Preparation of Recombinant Protein and Enzyme 18325633 Activity AssayThe coding sequence of human SSAT1, zebrafish ssat1 homologues, and the integrin a9 cytosolic, and hif-1a PAS-B domains were amplified (Table S1) from the cDNA of HEK293T cells or zebrafish embryos. PCR products were inserted into the EcoRI and XhoI sites of pET28a or pGEX2T and transformed into E. coli BL21 to express recombinant proteins with N-terminal His6tag or GST fusions. Following cell lysis, the His-tagged and GSTfused enzymes were purified by affinity chromatography on a HisTrap FF column (GE Healthcare) and a GSTrap FF column (GE Healthcare), respectively. The SSAT1 activity assay was described in our previous work [30].Cell Culture, Gene Transfection, and Cellular Protein ExtractionZebrafish ZF4 cells were cultured in 45 DMEM (Gibco), 45 Ham’s F12 medium (Gibco), and 10 FBS (Gibco) at 28uC with humidified air/CO2 (19:1 v/v). Human HEK293T cells (CRL11268) were cultured in DMEM with 10 FBS at 37uC with humidified air/CO2 (19:1 v/v). The HEK293T cells were transfected by using standard calcium phosphate precipitation and transfection of ZF4 cells was performed as described previously [30]. To prepare cellular proteins for analysis, cells were collected by centrifugation at 3006g, washed twice with PBS, and extracted with M-PER reagent (Thermo Scientific) according to the RE-640 site manufacturer’s instructions. The crude extract was dialyzed against 50 mM Tris-HCl (pH 7.5) with 16 Protease inhibitor (complete protease inhibitor cocktail, Roche) with an Amicon ultra centrifugal filter device (Millipore).Reverse Transcription (RT)-PCR and Molecular CloningTotal RNA was isolated from human HEK293T cells (CRL11268), adult zebrafish, and embryos with TRIzol reagent (Invitrogen). After DNase treatment, total RNA (500 ng) was reverse-transcribed to single-strand cDNA using SuperScript II reverse transcriptase (Clontech) according to the manufacturer’s instructions. Transcription of each gene was detected by RT-PCR with specific primers (see Table S1 in the supplemental material). The PCR reaction was initiated with DreamTaq (Fermentas) according to the manufacturer’s protocol. The reactions were separated by 1 agarose gel electrophoresis and visualized by staining with ethidium bromide. To prepare expression plasmids for zebrafish ZF4 (ATCC CRL2050) or HEK293T cells, the full-length ORF of human SSAT1, zebrafish ssat1a, ssat1b, and ssat1c, or DNA fragments encoding the cytosolic 4-IBP domain of zebrafish integrin 9a and the PAS-B domain o.He alignment is available upon request. Phylogenetic trees were constructed by the neighbor joining (NJ) method using the PHYLIP package [28] with the substitution model and represented with the FigTree software (http://tree.bio.ed.ac.uk/software/figtree/). Tree topology was evaluated by bootstrap resampling with 1000 replicates.the DpnI-resistant chimeric DNA was recovered by transformation into competent bacteria. The chimeric gene was named ssat1a248b, as it contains nucleotides 1?48 of ssat1a and the remainder of ssat1b. Chimeric genes ssat1a332b, ssat1a374b, ssat1a453b, ssat1b248a, ssat1b332a, ssat1b389a, and ssat1b467a were also prepared in this manner (Fig. 2B). The chimeric gene ssat1aba was prepared by using nucleotides 389?13 of ssat1a as the megaprimer and a plasmid containing ssat1a332b as template. Therefore, ssat1aba and ssat1bab contain most of the ssat1a and ssat1b sequences, but their 332?89 nucleotide regions are from ssat1b and ssat1a.Preparation of Recombinant Protein and Enzyme 18325633 Activity AssayThe coding sequence of human SSAT1, zebrafish ssat1 homologues, and the integrin a9 cytosolic, and hif-1a PAS-B domains were amplified (Table S1) from the cDNA of HEK293T cells or zebrafish embryos. PCR products were inserted into the EcoRI and XhoI sites of pET28a or pGEX2T and transformed into E. coli BL21 to express recombinant proteins with N-terminal His6tag or GST fusions. Following cell lysis, the His-tagged and GSTfused enzymes were purified by affinity chromatography on a HisTrap FF column (GE Healthcare) and a GSTrap FF column (GE Healthcare), respectively. The SSAT1 activity assay was described in our previous work [30].Cell Culture, Gene Transfection, and Cellular Protein ExtractionZebrafish ZF4 cells were cultured in 45 DMEM (Gibco), 45 Ham’s F12 medium (Gibco), and 10 FBS (Gibco) at 28uC with humidified air/CO2 (19:1 v/v). Human HEK293T cells (CRL11268) were cultured in DMEM with 10 FBS at 37uC with humidified air/CO2 (19:1 v/v). The HEK293T cells were transfected by using standard calcium phosphate precipitation and transfection of ZF4 cells was performed as described previously [30]. To prepare cellular proteins for analysis, cells were collected by centrifugation at 3006g, washed twice with PBS, and extracted with M-PER reagent (Thermo Scientific) according to the manufacturer’s instructions. The crude extract was dialyzed against 50 mM Tris-HCl (pH 7.5) with 16 Protease inhibitor (complete protease inhibitor cocktail, Roche) with an Amicon ultra centrifugal filter device (Millipore).Reverse Transcription (RT)-PCR and Molecular CloningTotal RNA was isolated from human HEK293T cells (CRL11268), adult zebrafish, and embryos with TRIzol reagent (Invitrogen). After DNase treatment, total RNA (500 ng) was reverse-transcribed to single-strand cDNA using SuperScript II reverse transcriptase (Clontech) according to the manufacturer’s instructions. Transcription of each gene was detected by RT-PCR with specific primers (see Table S1 in the supplemental material). The PCR reaction was initiated with DreamTaq (Fermentas) according to the manufacturer’s protocol. The reactions were separated by 1 agarose gel electrophoresis and visualized by staining with ethidium bromide. To prepare expression plasmids for zebrafish ZF4 (ATCC CRL2050) or HEK293T cells, the full-length ORF of human SSAT1, zebrafish ssat1a, ssat1b, and ssat1c, or DNA fragments encoding the cytosolic domain of zebrafish integrin 9a and the PAS-B domain o.
