Figure 2
Effect of Wnt3a point mutants on signaling activity. (A, C, E) Wnt3a point mutants from sites 1, 2 and 3 were tested in the Wnt reporter assay for their activity and compared with wild-type Wnt3a. HEK293T cells were transiently transfected with luciferase-based TCF/Wnt reporter with wild-type Wnt3a or the indicated mutant constructs. (B, D, F) Secretion levels of Wnt3a mutants analyzed by Western blot. HEK293T cells were transfected with wild-type Wnt3a or the indicated mutant constructs. Supernatants and cell lysates were analyzed 48 hr after transfection. Membranes were probed with Wnt3a and α-tubulin (loading control) antibodies. (G) Effect of double mutants on signaling activity. (H) Secretion levels of Wnt3a double mutants in HEK293T cells. Experiments were carried out in triplicate. Error bars depict standard deviation. Statistical significance in relative luciferase activity levels (A, C, E, G) of Wnt3a mutants compared with wild-type Wnt3a is indicated in the following way: *P < 0.05, **P < 0.01, ***P < 0.001 and n.s. as not significant according to Student’s t-test.