AnxA6 interferes with plasma membrane association of Dvl1 in a Ca²⁺-dependent manner.

<p><b>A:</b> Immunoblot analysis of the plasma membrane fraction isolated from WT and AnxA6-/- mouse articular chondrocytes treated with Wnt3a for the time periods indicated, or treated with the intracellular Ca²⁺ chelator, BAPTA-2AM followed by Wnt3a treatment, and analyzed with antibodies specific for Dvl1and AnxA6. Blots were also analyzed with antibodies specific for ATP1A1 (a plasma membrane protein) to control for equal loading. <b>B:</b> The optical densities of the Dvl1 and AnxA6 bands were quantitated by densitometry and normalized to the optical densities of the ATP1A1 bands. Results are expressed relative to the normalized optical densities of the Dvl1 or AnxA6 bands of vehicle-treated WT cells, which were set as 1. <b>C:</b> Immunoblot analysis of the nuclear fraction isolated from WT and AnxA6-/- mouse articular chondrocytes treated with Wnt3a for 30 min, or treated with the intracellular Ca²⁺ chelator, BAPTA-2AM followed by Wnt3a treatment, and analyzed with antibodies specific for ß-catenin. Blots were also analyzed with antibodies specific for lamin B (a nuclear protein) to control for equal loading. <b>D:</b> The optical densities of the ß-catenin bands were quantitated by densitometry and normalized to the optical densities of the lamin B bands. Results are expressed relative to the normalized optical densities of the ß-catenin bands of Wnt3a-treated WT cells, which were set as 1. The blots in <b>A</b> and <b>C</b> are representative of 3 separate experiments with similar results. The optical densities were analyzed on three different immunoblots, and values are mean ± SD. (*p < 0.05; **p < 0.01; ***p < 0.001).