Fluoxetine increases the ratio of phosphorylated β-catenin-to-β-actin, and enhances binding of Axin1 to β-catenin.

<p><b>(A-D)</b> Immunoblotting of total and phosphorylated β-catenin (β-cat) in differentiated ATDC5 cells treated with LiCl and fluoxetine for 48 hours. An antibody recognizes either total β-catenin or β-catenin phosphorylated at serine 33, serine 37, and threonine 41. Phosphorylation of β-cat at these residues triggers ubiquitination and degradation of β-cat [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184388#pone.0184388.ref007" target="_blank">7</a>]. The ratios of phosphorylated-to-total β-actin, total β-cat-to-total β-actin, and phosphorylated β-cat-to-total β-cat are normalized to those without LiCl or fluoxetine. The mean and SD in three independent experiments are indicated. <i>P</i>-values by one-way ANOVA are 0.00063 for phosphorylated β-cat-to-total β-actin <b>(B)</b>, 0.00091 for total β-cat-to-total β-actin <b>(C)</b> and 0.00083 for phosphorylated-to-total β-cat <b>(D)</b>. Statistical differences between 10 mM LiCl without fluoxetine (the second bar) and the others are indicated. *<i>p</i> < 0.05 and ***<i>p</i> < 0.005 by posthoc Tukey-Kramer test. <b>(E and F)</b> Immunoprecipitation of β-catenin, GSK3, and CK1 with anti-Axin1 antibody in whole lysates of L cells. The whole lysates were added with indicated concentrations of fluoxetine. Band intensities are normalized to that of Axin1, and also to that without fluoxetine. As a control, whole lysate is immunoprecipitated with normal mouse IgG. The mean and SD in three independent experiments are indicated. <i>P</i>-values by one-way ANOVA are 0.018 for β-catenin, 0.14 for GSK3, and 0.38 for CK1. *<i>p</i> < 0.05, **<i>p</i> < 0.01, and ***<i>p</i> < 0.005 by posthoc Tukey-Kramer test.