FIGURE 2.

The IWP compounds specifically inhibit Porcn acyltransferase activity. A, IWP2 inhibits Wnt fatty acylation. Cells transfected with an expression construct expressing either a fusion molecule consisting of Wnt3A and the Fc region of human IgG (Wnt3A-Fc) or IgG-Fc (Fc) alone are treated with C16 ω-alkynyl fatty acid (alkynyl-PA). Purified alkynyl-PA-labeled fusion protein bound to protein A-Sepharose is treated with biotin-azide reagent which enables protein detection using streptavidin-HRP. RNAi-mediated knock-down or overexpression of Porcn respectively results in loss or increase in Wnt3A-Fc protein labeling with alkynyl-PA. IWP2 is able to block the labeling of Wnt3A. B, IWP2 does not inhibit Hh fatty acylation. The same click chemistry strategy is used to monitor fatty acylation of Hh protein. C, IWP-Cy3 specifically binds to Porcn. COS-1 cells overexpressing Porcn or other members of the MBOAT family with recognized protein substrates (HHAT and GOAT) were treated with IWP-Cy3 and then gated for Cy3 staining. The number of IWP-Cy3-associated cells was scored as before.