Fig. 5.

Effect of inhibition of PKA and calcineurin pathways or mutagenesis of Ser²⁵⁶ in aquaporin-2 (AQP2) on the AQP2 shuttle. A: LLC-PK₁ cells stably expressing c-Myc-AQP2 (pseudo red) were preexposed to buffer, 0.3 µM myristoylated PKA inhibitor (mPKI), 50 µM st-Ht31, 50 µM st-Ht31-pro, or 1 µM CN-585 for 30 min at 37°C before exposure to buffer (a, c, e, g, and i) or 40 nM arginine-vasopressin (AVP; b, d, f, h, and j) for 20 min. Confocal images from fixed permeabilized cells were acquired as described in Fig. 1B. Scale bars = 5 μm. B: average ± SD values of AQP2 pixels outside a 300-nm partition of n = 30 control or AVP-exposed cells from 3 separate experiments. C: LLC-PK₁ cells transiently expressing wild-type (WT) c-Myc-AQP2 or the indicated point mutations in Ser²⁵⁶ or type-1 PDZ of c-Myc-AQP2 (pseudo red) were exposed to buffer (k, m, o, q, and s) or 40 nM AVP (l, n, p, r, and t) and processed as in A. D: average ± SD values of AQP2 pixels outside a 300-nm partition of n = 30 images/condition from n = 3 separate experiments. Statistical comparisons in B or D were carried out by one-way ANOVA followed by a Bonferroni’s test. NS indicates nonsignificant differences between the column pairs. ***P < 0.001.