AQP2 phosphorylation and coimmunoprecipitations.

LLC-PK₁ cells expressing wild-type (WT) AQP2 or AQP2∆PDZ were treated overnight with 50 µM indomethacin and then switched to Opti-MEM with 1% serum and 50 µM indomethacin for 6 h. Cells were exposed to buffer (control) or stimulated with 40 nM AVP or 20 µM forskolin (FSK) for 10 min. Indomethacin was included in all phosphorylation conditions to reduce basal cAMP and phospho-Ser²⁵⁶ AQP2 levels so that the effect of AVP or FSK on this parameter could be observed (10). Cells were lysed in 500 μl of 20 mM HEPES, 150 mM NaCl, 2 mM EDTA, 10% glycerol, 0.5% Triton X-100, and complete protease and phosphatase inhibitor cocktail (Roche, Branchburg, NJ). Lysates were rotated at 4°C for 1 h followed by centrifugation at 14,000 g at 4°C for 20 min. Soluble extracts were precleared by 30-min incubation with 30 μl protein G-Sepharose. After protein concentrations were equalized across all samples, lysates were added to ~5 μl of anti-c-Myc 9E-10 IgG agarose beads for 4 h at 4°C. Immune complexes were washed five times, and proteins were eluted in 40 μl of elution buffer (200 μg/ml c-Myc peptide, 20 mM HEPES, 50 mM NaCl, 0.1% cholesterol, and 8 mM EDTA). Eluted immune complexes were separated by SDS-PAGE and subjected to Western blot analysis with anti-phospho-Ser²⁵⁶ AQP2 antibody as previously described (43).

Coimmunoprecipitations between c-Myc-AQP2 and SAP97 were performed as follows. LLC-PK₁ cells expressing c-Myc-AQP2 were lysed, and insoluble cellular debris was removed by centrifugation. After protein concentrations were equalized across all samples, lysates were added to ~5 μl of anti-c-Myc agarose beads. Reverse experiments involved the addition of equal amounts of cell lysates to anti-SAP97 IgG bound to protein G-agarose beads (42). Control experiments were performed by incubating lysates with preimmune IgG conjugated to protein G-agarose or protein G-Sepharose beads at the same concentration for 4 h at 4°C. Immune complexes were washed five times in lysis buffer and eluted from the beads with 2× Laemmli sample buffer containing 40 mM dithiothreitol and subjected to Western blot analysis (43). To normalize for input protein levels, 5% of each cell lysate was subjected to Western blot analysis using anti-β-actin antibody. Luminescence was acquired using the Bio-Rad XRS chemiluminescence documentation system on short exposure blots, and densitometric changes were quantified as described above.