Purified Wnt5a Protein Inhibits Canonical Wnt Reporter Activation

<div><p>(A) Coomassie blue–stained gel showing the Wnt5a protein purification scheme.</p> <p>(B) The 24-h Wnt3a protein treatment activates the STF luciferase reporter in a dose-responsive manner.</p> <p>(C) Concomitant Wnt5a protein treatment inhibits Wnt3a-induced STF reporter activation.</p> <p>(D) Time-course analysis of Wnt5a-mediated inhibition of Wnt3a. At approximately 40 h post-transfection, cells were treated with Wnt3a protein for 0, 2, 4, 6, or 8 h alone or in conjunction with Wnt5a at <i>t</i> = 0, <i>t</i> = 1 h, or <i>t</i> = 3 h. (Filled diamonds indicate Wnt3a treatment alone; open diamonds indicate Wnt3a treatment at <i>t</i> = 0 plus Wnt5a protein treatment at <i>t</i> = 0; filled triangles indicate Wnt3a treatment at <i>t</i> = 0 plus Wnt5a protein treatment at <i>t</i> = 1; open circles indicate Wnt3a treatment at <i>t</i> = 0 plus Wnt5a protein treatment at <i>t</i> = 3.) </p> <p>(E) Wnt5a pretreatment does not enhance its inhibitory activity. At 24 h post-transfection, cells were pretreated with vehicle or Wnt5a protein (200 ng/ml) for 8 h. Cells were then treated with vehicle, Wnt3a alone, or Wnt3a concomitantly with Wnt5a for an additional 24 h, and luciferase assay was performed. (Filled squares indicate cells that were pretreated with vehicle for 8 h prior to Wnt addition; open squares indicate cells pretreated with Wnt5a protein for 8 h prior to Wnt addition.) Unless stated in text, luciferase activity was measured 48 h after transfection, approximately 24 h after Wnt treatment, and results are shown as the average fold change ± SD in luciferase activity over transfected, untreated negative controls.</p>