Journal: American Journal of Physiology - Renal Physiology
Article Title: Activation of the metabolic sensor AMP-activated protein kinase inhibits aquaporin-2 function in kidney principal cells
Figure Lengend Snippet: Changes in AQP2 phosphorylation pattern following dDAVP treatment in the presence or absence of AMPK activator in mpkCCD c14 cells. Twenty-four hours posttransfection with wild-type AQP2 plasmid, cells were incubated in the presence or absence of AICAR (2 mM) for 4 h, and then dDAVP (10 nM) or vehicle was added for 30 min before cell harvesting. Immunoblot analysis of immunoprecipitated V5-AQP2 was performed using the corresponding rabbit polyclonal antibodies against phosphorylated AQP2 COOH-terminal sites: −S256, −S261, −S264, and −S269. To confirm successful immunoprecipitation, the membranes were reblotted using anti-AQP2 and anti-V5 antibodies coupled to HRP. A : representative set of immunoblots from these experiments using antibodies against AQP2 and its different COOH-terminal phosphorylation sites. B : quantification of AQP2 phosphorylation signal for different AQP2 COOH-terminal sites normalized for protein loading, as assessed by densitometry of the immunoblot. Values are means ± SE of 4 independent experiments. * P
Article Snippet: After each incubation of the membrane with a particular anti-phospho COOH-terminal antibody, the nitrocellulose membrane was stripped and reblotted using anti-AQP2 antibody (Alomone Labs).
Techniques: Plasmid Preparation, Incubation, Cell Harvesting, Immunoprecipitation, Western Blot