[PMC free article] [PubMed] [CrossRef] [Google Scholar] 24

[PMC free article] [PubMed] [CrossRef] [Google Scholar] 24. SAP97 overlapped in the kidneys and LLC-PK1 cells and that knockdown of SAP97 inhibited the translocation of AQP2 in response to AVP. Binding between AQP2 and SAP97 was mediated by specific interactions between the second PDZ of SAP97 and PBM of AQP2. Mechanistically, inactivation of the PBM of AQP2, global delocalization of PKA, or knockdown of SAP97 inhibited AQP2 translocation as well as AVP- and forskolin-mediated phosphorylation of Ser256 in AQP2, which serves as the major translocation barcode of AQP2. These results suggest that the targeting of PKA to the microdomain of AQP2 via SAP97-AQP2 interactions in association with cross-talk between two barcodes in AQP2, namely, the PBM and phospho-Ser256, plays an important role in the translocation of AQP2 in the kidney. 30). These data are presented as means??SD. For colocalization between SAP97 and AQP2, LLC-PK1 cells were transfected with 1.5 g of plasmids harboring SAP97-GFP (12) Necrostatin 2 and c-Myc-AQP2 for 16 h, switched to Opti-MEM with 1% serum for 6 h, and then fixed. Cells were permeabilized, blocked, Rabbit Polyclonal to MRPL16 and incubated with CF555-anti-c-Myc and visualized using CF555/Cy3 (pseudo red for AQP2) and GFP (pseudo green for SAP97) laser settings. Cell surface biotinylation and glutathione-S-transferase pulldown assays. LLC-PK1 cells on 15-cm culture dishes were exposed to buffer or 40 nM Necrostatin 2 AVP Necrostatin 2 for 20 min at 37C. The plates were quickly washed free of culture media and reagents with ice-cold HBSS and then incubated in a total volume of 5 ml HBSS supplemented with 3 mg/ml of the cell-impermeant biotin-amidohexanoic acid 3-sulfo-= 5 separate experiments were calculated and keyed into the statistical software program to estimate the average fold increase??SD for each condition. The creation of individual SAP97 PDZs fused to glutathione-at 4C for 20 min. Soluble extracts were precleared by 30-min incubation with 30 l protein G-Sepharose. After protein concentrations were equalized across all samples, lysates were added to ~5 l of anti-c-Myc 9E-10 IgG agarose beads for 4 h at 4C. Immune complexes were washed five times, and proteins were eluted in 40 l of elution buffer (200 g/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 Necrostatin 2 all samples, lysates were added to ~5 l of anti-c-Myc agarose beads. Reverse experiments involved the addition of equal amounts of cell Necrostatin 2 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 4C. 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–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. Animals and immunohistochemistry. Animal experiments were performed according to protocols approved by the Institutional Animal Care and Use Committees of the University of Louisiana-Monroe and University of Tennessee Health Sciences Center. Experiments conformed with the NIH (NIH Pub. No. 85-23, Revised 1996). Eight-week-old male C57BL/6NTac mice (weighing between 24 and 28 g) were obtained from Taconic. Mice (= 4) were euthanized by barbiturate overdose (pentobarbital, 150 mg/kg ip), and their.