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| Peptide Synthesis | Rabbit polyclonal antibody was raised against aa 35–48 (DGKSHGHDGHDGQS) of the translated amino acid sequence of Rhag, against aa 449–462 (LASVRTEESEKLNS) of the translated amino acid sequence of Rhbg and against aa 388–401 (LEGNYANRGAGTQG) of the translated amino acid sequence of Rhcg2 (GenScript, Piscataway, NJ, USA). The anti-Rhag (10 µg), anti Journal of Experimental Biology • Advance article Rhbg (0.17 µg) and anti-Rhcg2 (16.65 µg) antibodies were pre-incubated with the immunizing peptide of Rhag (50 µg), Rhbg (1.7 µg) or Rhcg2 (83.25 µg) provided by Genscript, respectively, in a total volume of 200 µl at 25°C for 1 h. The 1-Step Human Coupled IVT KitDNA (Thermo Fisher Scientific)was used to synthesize the recombinant protein of Rhcg1 from 1 µg of the complete coding cDNA sequence of rhcg1, following the manufacturer’s instruction. Sections were double labelled using: (1) anti-Nkaα1c rabbit polyclonal antibody (Genscript)with anti-Nkaα1a mouse monoclonal antibody (Abmart Inc., Shanghai, China; Ching et al.2013) to demonstrate that Nkaα1c was not expressed in ionocytes of the freshwater control, (2) anti-NKAαRb1 rabbit polyclonal antibody [a pan-specific antibody originally designed by Ura et al. (1996)] with anti-Nkaα1b mouse monoclonal antibody (Abmart Inc.; Ching et al. 2013) to demonstrate that Nkaα1b was not expressed in ionocytes in fish exposed to ammonia, (3) anti-Nkaα1a mouse monoclonal antibody or anti-Nkaα1c mouse polyclonal antibody (Genscript) with anti-Rhag, anti-Rhbg, anti-Rhcg1 or anti-Rhcg2 rabbit polyclonal antibodies to elucidate the type of Rhgp associated with the ammonia-type ionocytes in fish exposed to ammonia, and (4) anti-Cftr mouse monoclonal antibody (Clone #24-1; R&D systems, Minneapolis, MN, USA) with anti-Rhag to demonstrate that they can be colocalized to the apical membrane of the same ionocyte in fish exposed to ammonia. | Get A Quote |
The freshwater climbing perch， ， is an obligate air-breathing and euryhaline teleost capable of active ammonia excretion and tolerant of high concentrations of environmental ammonia. As Rhesus glycoproteins (RhGP/Rhgp) are known to transport ammonia， this study aimed to obtain the complete cDNA coding sequences of various isoforms from the gills of ， and to determine their mRNA and protein expression levels during 6 days of exposure to 100 mmol l NHCl. The subcellular localization of Rhgp isoforms in the branchial epithelium was also examined in order to elucidate the type of ionocyte involved in active ammonia excretion. Four (， ， and ) had been identified from the gills of They had conse... More
