Figure 6.

Sipa1l1 preferentially interacted with Ser-269 non-phosphorylated AQP2. A, biotin-labeled synthetic AQP2 COOH-terminal peptides. B and C, representative immunoblot (IB) and summary of Sipa1l1 interaction with Ser-256 phosphorylated versus Ser-256 and Ser-269 doubly phosphorylated AQP2 peptide. The mpkCCD cell lysate was incubated with the biotin-labeled AQP2 peptides. After binding and washes, the eluate was detected for Sipa1l1 with immunoblotting. Numbers are signal intensities (means ± S.D.). Asterisk indicates significance, p < 0.05, t test. D and E, representative immunoblot and summary of Sipa1l1 interaction with S269A versus S269D AQP2 protein. The cell lysates from S269A or S269D AQP2 expressing mpkCCD cells were immunoprecipitated (IP) with the AQP2 antibody before immunoblotting for Sipa1l1. F and G, immunofluorescence staining for S269D and S269A AQP2 in control and Sipa1l1 knockdown mpkCCD cells in the absence of dDAVP.