Figure 5. Wnt1 S224 is more critical than C93 for ß-catenin dependent signaling.

Panel A: HEK293T cells were transfected with 8XSuperTop/FopFlash [64], renilla luciferase, and indicated pcDNA expression constructs. DNA concentrations were held constant within each series of experiments. Values shown reflect the experimental data (Firefly/Renilla) minus FopFlash (Firefly/Renilla). The relative luciferase activity of wild-type Wnt1/Wnt3a was set at 1. Single and double Wnt1/Wnt3a mutants show significantly less activity than wild-type Wnts (p<1×10⁻¹⁰). Differences between C93S and C93A mutants are not significant nor are those between S224A and C93A/S224A. Error bars indicate standard error for at least 15 data points from 3 independent replicates. Panel B: HEK293T cells were transfected as in Panel A, lysed, subjected to SDS-PAGE, and blotted onto PVDF. Blots were probed with anti-Wnt1 or anti-ß-tubulin. Panel C: The Western blots were scanned and analyzed using NIH ImageJ. This experiment was performed 3 times. Error bars represent +/− standard error. Panel D: LS/L cells were then transiently transfected with constructs encoding GFP or wild-type/mutant Wnt1. As before, the relative luciferase activity of wild-type Wnt1 was set at 1. Wnt1 and Wnt1C93A show statistically significant increases in reporter activity as compared to GFP (p<1×10⁻⁷) while Wnt1S224A and Wnt1C93AS224A do not. Error bars represent standard error from at least 3 independent replicates with a total of 12 data points.