| Products/Services Used | Details | Operation |
|---|---|---|
| Peptide Synthesis | A rabbit polyclonal anti-Nhe3 antibody was designed against residues 736–749 (GKSPDRSRSYHSGD) of the deduced Nhe3 sequence from A. testudineus (GenScript, Piscataway, NJ, USA). Anti-Nhe3 (10µg) was pre-incubated with the immunizing peptide of Nhe3 (150 µg) in a final volume of 10 ml at 25◦C for 1 h to validate the specificity of the antibody. Antigen retrieval was carried out with 0.05% citraconic anhydride in a boiling water bath (Namimatsu et al., 2005). After blocking with 5% BSA/TPBS, sections were incubated with: (1) the rabbit anti-NKAαRb1 polyclonal antibody (a pan-specific antibody originally designed by Ura et al., 1996 for labeling Nka αsubunits) co-labeled with the mouse anti-Nkaα1b monoclonal antibody (Abmart, Shanghai, China; Ching et al., 2013) or the mouse anti-Nkaα1c polyclonal antibody (Genscript; Chen et al., 2017) to examine whether Nkaα1b and Nkaα1c were present in different types of ionocyte, (2) the custom-made rabbit antiNhe3 antibody (Genscript) co-labeled with the mouse antiNkaα1a monoclonal antibody (Abmart), or the mouse antiNkaα1b monoclonal antibody (Abmart), or the mouse antiNkaα1c polyclonal antibody (Genscript) to determine the type of Nka α-isoform associated with Nhe3 in the gills of A. testudineus, and (3) the mouse anti-Nkaα1b antibody (Abmart) or the mouse anti-Nkaα1c antibody (Genscript) co-labeled with the rabbit anti-Rhcg1 antibody (kindly provided by S. Hirose) or the rabbit anti-Rhcg2 antibody (Genscript; Chen et al., 2017) to examine if they were localized to the same type of ionocyte.To validate the specificity of anti-Nkaα1c binding in this study, anti-Nkaα1c (6.25 µg) was pre-incubated with the immunizing peptide of Nkaα1c (62.5 µg) provided by Genscript, in a total volume of 10 ml at 25◦C for 1 h before performing Western blotting and the results are shown in Supplementary Figure S1. | Get A Quote |
The freshwater climbing perch, Anabas testudineus, is an euryhaline teleost and an obligate air-breather with the ability to actively excrete ammonia. Members of the Na+/H+ exchanger (NHE) family help maintain intracellular pH homeostasis and ionic balance through the electroneutral exchange of Na+ and H+. This study aimed to obtain, from the gills of A. testudineus, the full cDNA coding sequence of nhe3, and to determine the effects of exposure to seawater or 100 mmol l−1 of NH4Cl in fresh water on its mRNA and protein expression levels. Efforts were also made to elucidate the type of ionocyte that Nhe3 was associated with in the branchial epithelium of A. testudineus. The transcript level and protein abunda... More
