Classical Wnt/Frizzled/ß-catenin

<p><b>signaling is not involved in US28-mediated Tcf-Lef activation.</b> A, Western blot analysis of total cell extracts of NIH-3T3 cells, stably expressing US28 or an empty plasmid (mock) which were treated with Wnt3a- (overnight, 200 ng/ml) and vehicle-treated mock cells. The blot was probed with antibodies recognizing the non-phosphorylated (active ß-catenin, total ß-catenin and actin. A representative blot is shown and normalized quantifications of (active) ß-catenin of independent experiments are shown below the blot. B, Western blot analysis of total cell extracts of NIH-3T3 cells stably expressing US28, the non G-protein coupling US28 mutant R¹²⁹A or an empty plasmid (mock) and Wnt3a-treated mock cells. The blot was probed with antibodies recognizing Lrp6-phospho-ser¹⁴⁹⁰ and actin. A representative blot is shown and normalized quantifications of Lrp6-phospho-ser¹⁴⁹⁰ of independent experiments are shown below the blot. C, HEK293T cells co-transfected with the Tcf-Lef reporter gene construct and either US28-expressing or an empty control plasmid (mock) exposed to Wnt3a (overnight, 200 ng/ml). Luciferase activity was measured 24 hr after transfection and is displayed here as the percentage of the non-treated mock control that is set at 100%. D, HEK293T cells co- transfected with the Tcf-Lef reportergene and an US28-expressing construct or empty plasmid control (mock) were exposed to various concentrations (ON, 10–25 µM) of the COX2 inhibitor celecoxib (Cxb). Tcf-Lef reporter gene activation was measured 24 hr after transfection and is displayed here as the percentage of the mock control that is set at 100%.