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.