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First published online November 14, 2008
Journal of Experimental Biology 211, 3750-3758 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.018440
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Branchial FXYD protein expression in response to salinity change and its interaction with Na+/K+-ATPase of the euryhaline teleost Tetraodon nigroviridis

Pei-Jen Wang*, Chia-Hao Lin*,{dagger}, Hau-Hsuan Hwang and Tsung-Han Lee{ddagger}

Department of Life Sciences, National Chung-Hsing University, Taichung 402, Taiwan


Figure 1
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  		Fig. 1. (A) Pufferfish FXYD (pFXYD) nucleotide sequence and deduced amino acid sequence. The transmembrane domain is shaded gray; the predicted signal peptide is underlined; the circle indicates the site for phosphorylation; the boxes indicate the highly similar FXYD motif and two glycine residues (G39 and G50). (B) Phylogenetic tree of pFXYD with known human FXYD members and related EST sequences from various teleost fish. Accession numbers are: shark PLMS, CAD88978; human FXYD1, NP_005022; human FXYD2.a, NP_001671; human FXYD2.b, NP_067614; human FXYD3, NP_005962; human FXYD4, NP_775183; human FXYD5, NP_054883; human FXYD6, NP_071286; human FXYD7, NP_071289; zebrafish EST-FXYD6, AW153757; zebrafish EST-FXYD8, AI958251; zebrafish EST-FXYD9, AW455046; medaka EST-FXYD.a, AU169681; medaka EST-FXYD.b, AU169966; Fugu EST-FXYDa, CA332188; Xenopus FXYD1, NP_001011320.

 

Figure 2
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  		Fig. 2. RT-PCR analysis showing expression of pFXYD in various tissues of fresh water (FW)- and seawater (SW)-acclimated pufferfish. The pFXYD gene was found in the gill, kidney, gut, liver, eye, brain, muscle and heart of the pufferfish acclimated to fresh water (FW) or seawater (SW). β-Actin was used as an internal control.

 

Figure 3
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  		Fig. 3. Quantification of relative mRNA abundance of branchial pFXYD. (A) RT-PCR analysis confirmed the primer specificity. M, markers. (B) Comparison between branchial mRNA abundance of freshwater (FW)- and seawater (SW)-acclimated pufferfish, as quantified by real-time PCR. β-Actin was used as an internal control. Values are means ± s.e.m. (N=6). The asterisk indicates a significant difference between the FW and SW groups (P<0.05, Student's t-test).

 

Figure 4
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  		Fig. 4. (A) Specificity analysis of the pFXYD antiserum raised against a synthetic peptide corresponding to the N-terminal region of pufferfish FXYD. Total gill lysates were separated by SDS-polyacrylamide gel electrophoresis and transferred to PVDF membrane. A single immunoreactive band was detected in both freshwater and seawater gill samples by the HRP detection system. The pre-immune serum was used as the negative control, which revealed no immunoreactive band. (B) Membrane fractions had an immunoreactive band at 13 kDa. β-Actin was used as the loading control. (C) The relative abundance of the pFXYD protein expressed in gills of pufferfish acclimated to fresh water (FW) was significantly higher than in the SW group (N=6). a.u., arbitrary units. The asterisk indicates a significant difference (P<0.05, Student's t-test). Values are means ± s.e.m.

 

Figure 5
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  		Fig. 5. Immunolocalization of pFXYD protein (red; A and D) and Na+/K+-ATPase (NKA; green; B and E) in frozen longitudinal sections of gills of pufferfish acclimated to fresh water (FW) or seawater (SW). After double staining of the same sections, merged confocal microscope images revealed that NKA and pFXYD are colocalized (yellow in C and F) in the basolateral membrane of epithelial cells in gill filaments. F, filament. Scale bar, 50 µm.

 

Figure 6
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  		Fig. 6. Co-immunoprecipitation of Na+/K+-ATPase (NKA) with pFXYD protein (A), and pFXYD with NKA (B). pFXYD and NKA were immunoprecipitated from gill total lysates of freshwater pufferfish by primary antibodies, then the immune complexes were analyzed by SDS-PAGE and subsequent immunoblotting for NKA and pFXYD protein, respectively. Immunoreactive bands of NKA and FXYD were detected at 100 kDa (A) and 13 kDa (B), respectively. In B, the 55 kDa band in lane 3 is the IgG heavy chain of pFXYD antibody. M, markers; lane 1, western blot detection of the opposite antibody (experimental group); lane 2, negative control; lane 3, positive control of immunoblot using the same antibody with immunoprecipitation.

 

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© The Company of Biologists Ltd 2008