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. 2000 Nov 13;151(4):919–930. doi: 10.1083/jcb.151.4.919

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© 2000 The Rockefeller University Press

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/).

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Phosphorylation of AQP2 in oocytes. Oocytes, injected with wt-AQP2 or AQP2-S256A cRNA, were labeled with [³²P]orthophosphate for 2 d. Subsequently, oocytes were not stimulated (ctrl, AQP2-S256A), or were incubated with 5 × 10⁻⁴ M 8-Br-cAMP or 10⁻⁴ M H89 for the indicated time periods. Total membranes were isolated and divided into two portions. One portion was solubilized, AQP2 was immunoprecipitated and subjected to PAGE, the gel was dried, and labeled AQP2 (p-AQP2) was visualized using autoradiography. The other portion was directly immunoblotted for AQP2 (Total AQP2).