FIGURE SUMMARY
Title

Differences in a Single Extracellular Residue Underlie Adhesive Functions of Two Zebrafish Aqp0s

Authors
Vorontsova, I., Hall, J.E., Schilling, T.F., Nagai, N., Nakazawa, Y.
Source
Full text @ Cells
Figure 1

Zebrafish Aqp0b has adhesive properties. (A) Adherent cell percentages are shown for homotypic pairs of stable cell lines expressing positive control rat AQP0 (rAQP0), negative control rat AQP1 (rAQP1), zebrafish Aqp0a or Aqp0b, as well as cells transfected with an empty vector (L-(−)). Each lane represents n = 6–8. (B) Similar expression levels of rat AQP0 and zebrafish Aqp0 proteins shown by Western blotting using an anti-human C-terminus AQP0 antibody, and an anti-β-actin antibody as the loading control. Sample blot, top panel; quantification, bottom panel (n ≥ 3). (C) Heterotypic adhesion tested between zebrafish orthologs. Aqp0 plated first is indicated at the bottom. Statistical differences are shown as * having p < 0.05 or as ** having p < 0.01.
Figure 2

Zoo protein alignments of AQP0 extracellular loops. Amino acid alignment of extracellular loops A, C, and E of AQP0 across selected mammalian and piscine species. RasMol coloring. Human (Homo sapiens), bovine (Bos taurus), mouse (Mus musculus), and rat (Rattus norvegicus) AQP0 (also known as MIP) are shown. The fish Aqp0s shown include MIPfun of killifish (Fundulus heteroclitus) and duplicated Aqp0s for zebrafish (Danio rerio) and Atlantic salmon (Salmo salar). Two amino acids of interest indicated as boxed sites 110 and 195 were mutated in this study.
Figure 3

Mutational analysis reveals the importance of site 110 for adhesion of Aqp0b. (A) Adhesive properties were tested between homotypic pairs of stable cell lines expressing WT and mutant variants of Aqp0a and Aqp0b. Each lane represents n = 6–8. (B) Western blot analysis example showing equal expression of the protein between Aqp0 WT and mutant variants using zebrafish specific rabbit anti-Aqp0a and anti-Aqp0b sera quantified in (C) (n ≥ 3). Statistical differences are shown as ** having p < 0.01.
Figure 4

MIPfun’s adhesive properties are enhanced by an N110P mutation. (A) Adhesive properties were tested between homotypic pairs of stable cell lines expressing WT MIPfun and mutated variants. Each lane represents n = 6–8. (B) Western blot analysis example shows equal expression of the protein between WT and mutant variants using the rabbit anti-zebrafish Aqp0b sera quantified in (C) (n ≥ 3). Statistical differences are shown as ** having p < 0.01.
Acknowledgments
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