Computational protocol: TGF-β1 Exerts Opposing Effects on Grass Carp Leukocytes: Implication in Teleost Immunity, Receptor Signaling and Potential Self-Regulatory Mechanisms

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[…] Five µg of total RNA was extracted from grass carp HKL using TRIzol Reagent (Invitrogen) and reverse-transcribed into cDNA using Superscript III reverse transcriptase (Invitrogen). The DNA fragment for Alk5 was obtained by PCR amplification with the degenerated primers (alkF and alkR, ) and subcloned into pGM-T vector (TaKaRa, Dalian, CH) for sequencing. Based on the partial sequences, gene-specific primers (alk5p1, alk5p2, alk3p1 and alk3p2) were designed (), and 3′/5′-RACE were performed using the GeneRacer Kit (Invitrogen). The PCR products were gel-purified and subcloned into pGM-T vector for DNA sequencing. Both the open reading frame and the deduced protein sequence of Alk5 were analyzed by ORF Finder (http://www.ncbi.nlm.nih.gov/projects/gorf/). Following the similarity analysis by using nucleotide and protein BLAST programs from NCBI, multiple alignments were carried out by using DNAMAN software (Lynnon Biosoft, Quebec, CA). Characteristic motifs were predicted by ScanProsite (http://www.expasy.ch/tools/scanprosite/). Phylogenetic trees were constructed using the Neighbor-Joining method by Molecular Evolutionary Genetics Analysis (MEGA) 3.1 with bootstrap value of 1,000. […]

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