Fig. 4. Effect of apelin-13 on AQP-2 protein expression. (A) Representative immunoblot of five independent experiments showing the expression of non-glycosylated and glycosylated forms of AQP2 in mpkCCD cells treated or not with dDAVP (10 nM, 24 h) in the absence or presence of apelin-13 (200 nM) for 8 h or 24 h. β-actin was used as a loading control. (B) Densitometric analysis of AQP2 expression. §§§ p<0.001, ** p<0.01, compared to dDAVP treatment (5 independent experiments; two-way ANOVA with a posthoc Bonferroni multiple comparison test). (C) Representative immunoblot of five independent experiments performed with protein extract samples treated (+) or not (-) with 100U of peptide-N-glycosidase F for 1 hour showing that the diffuse band corresponds to the glycosylated AQP-2 protein form and not that of other non-specific binding. In these experiments, mpkCCD cells were grown and treated with dDAVP as described above (10 nM, 24 h) and treated in the absence (-) or presence of apelin-13 for 8 h (+). g-AQP2: glycosylated aquaporin, ng-AQP2: non glycosylated aquaporin.