Figure 1.

Active WNT3A Molecules Can Be Stably Immobilized onto Aldehyde-Coated Surfaces and Induce WNT/β-Catenin Signaling

(A) Diagram of the one-step chemical reaction to immobilize WNT3A onto an aldehyde-functionalized surface.

(B) Representative stain-free gel (left) and αWNT3A immunoblot (right) of immobilization process showing WNT3A input (10 ng), unbound WNT3A collected after the incubation and three PBS washings (W1, W2, W3) of the surface. 0.1% BSA alone was also loaded on the gel as a control.

(C) Immunoblot of αWNT3A including wash steps after immobilization of WNT3A along with media supernatant after surfaces incubated at 37°C for 7 hr with (left) and without (right) serum. Proteins were precipitated with trichloroacetic acid before loading onto electrophoresis gels. Long exposure (below) included for visualizing low protein levels.

(D) Normalized luciferase activity assay (reported as fold activation) using LS/L cells harboring a 7xTCF-luciferase reporter. Cells seeded onto immobilized WNT3A surfaces at various concentrations with or without inactivation of WNT3A (DTT treatment).

(E) Luciferase activity assay of WNT3A immobilization conditions: BSA (washed with PBS post-immobilization), WNT3A (immobilized in 1% Chaps solution), WNT3A (washed with 1% Chaps solution post-immobilization), WNT3A (washed with PBS post-immobilization) and WNT3A in solution (50 ng/ml).

(F) Surfaces immunostained for αWNT3A. The scale bar represents 10 μm.

(G) Luciferase activity assay comparing WNT3A immobilization onto different surfaces: glass (washed with 1% Chaps solution post-immobilization procedure), glass (washed with PBS), aldehyde (pre-blocked with laminin protein), and aldehyde (washed with PBS).

(H and I) Luciferase activity assay comparing freshly immobilized surfaces to surfaces stored at 4°C for 1 week and 21 days.

n = 3 independent experiments, mean ± SD; statistical significance using a one-way ANOVA; ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001; ns, not significant.