FIGURE 4.

Mutational analysis of conserved residues surrounding Ser-209 in Wnt3a. A, multiple sequence alignment of a highly conserved region of Wnt family members from diverse species. Black boxes denote residues that are conserved in 80% or more sequences. B, COS-1 cells transfected with FLAG-Porcn and the indicated Wnt3a constructs were labeled with ¹²⁵I-IC15:1 for 5 h. Cell lysates were immunoprecipitated (IP) with anti-Myc or anti-FLAG antibodies and analyzed by SDS-PAGE and phosphorimaging (top panel) or Western blotting (WB, middle and bottom panels) with anti-Wnt3a or anti-FLAG antibodies. The experiment was repeated three times in duplicate; a representative image is shown. C, black bars, quantification of experiments in B. Phosphorimaging signals were normalized to Wnt3a protein levels and then expressed as a percentage of WT Wnt3a (set at 100%). Each bar represents the mean ± S.D. (n = 3–6). Gray patterned bars, HEK293T cells were transfected with STF or FOPFlash, RL, and either WT or mutant Wnt3a. 48 h after transfection, STF luciferase activity was measured, normalized to RL and FOPFlash activity, and expressed as a percentage of WT; bars represent means ± S.E. (n = 3). D, lysates from COS-1 cells co-expressing WT Porcn and the indicated Wnt3a constructs were immunoprecipitated with anti-Myc or anti-FLAG antibody followed by Western blotting with anti-Wnt3a or anti-FLAG antibody. E. V., empty vector.