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. 2008 Sep 17;295(6):C1476–C1487. doi: 10.1152/ajpcell.00344.2008

Fig. 6.

Fig. 6.

Role of S256 phosphorylation in constitutive and VP-induced AQP2 exocytosis. A: 15 min of preincubation with the PKA inhibitor H-89 blocked VP/FK-induced burst of exocytosis in AQP2-expressing cells. Extracellular medium fluorescence after 15 min was background subtracted and normalized to LLC-AQP2-ssYFP control. B: Western blot analysis shows similar expression of AQP2 (left, lanes 2 and 4) and ssYFP (right, lanes 6 and 8) in wild-type AQP2 and AQP2-S256A-expressing cells. Results of 3 independent blots were quantified by densitometry and shown as ratio of ssYFP or AQP2 to actin-loading controls (left) normalized to LLC-AQP2-ssYFP expression. C: constitutive exocytosis is reduced in LLC-AQP2-S256A-ssYFP cells. Although absolute value of the response to VP/FK was reduced compared with wild-type controls (LLC-AQP2-ssYFP), fold increase in ssYFP secretion was greater in the S256A mutant cell line. Relative fluorescence values were background subtracted and calculated relative to LLC-AQP2-ssYFP at 15 min. D: real-time PCR quantification of mRNA reveals 0.5-fold higher expression of AQP2-S256A than ssYFP (see Fig. 3B) and similar ssYFP expression levels in ssYFP and S256-expressing cell lines. P0 mRNA was used as an internal control, and all values were normalized to corresponding LLC-AQP2-ssYFP levels (set at 100%). *P < 0.05. **P < 0.01.