Fig. 1. RNF43 function requires phosphorylation of a serine triplet.
a–c Regulation of Wnt3a-mediated activation of Wnt/β-catenin signalling by RNF43 mutants was examined using SuperTopFlash (STF)-luciferase reporter assays. Luciferase activity in empty vector-transfected negative control (NC) cells was set to 1. SRR: serine rich region. Characters shown in red indicate amino acids after substitution (b, c). PA: protease-associated domain. TM: transmembrane region. RING: RING finger domain. d Expression of frizzled (Fzd) at the surface of RNF43 mutant-expressing cells was measured via flow cytometric analysis. FACS data was acquired and displayed with same strategy shown in Supplementary Fig. 1g. Grey or black lines, or grey fills indicate not stained, RNF43 stably expressed or mock cells, respectively. Expression in mock-transfected cells was set to 1. e Cellular phosphorylation of RNF43 was examined using 32Pi metabolic labelling. Radio-labelled RNF43 levels were normalised to total RNF43 protein levels. The relative phospho-RNF43 level in mock-transfected cells was set to 1. Bar graphs and error bars in this figure represent mean ± standard deviation (sd) of at least three biologically independent experiments. Red circles indicate individual values of each sample. The P values for the indicated comparisons were determined by one-way analysis of variance (ANOVA) (P < 0.05). n = 3 (a–d), n = 4 (e) biologically independent samples. Asterisks or ND indicates significant or no significant difference in indicated comparisons, respectively.