FIGURE 5:

PrP^c interaction with the transcription factor TCF7L2 occurs mainly via the β-catenin–binding domain of TCF7L2. (A) Schematic diagram of the human TCF7L2 protein and the full-length TCF7L2 tagged with the FLAG octapeptide (FLAG-TCF7L2^FL) construct. SW480 cells were transfected with FLAG-TCF7L2 ^FL. Transfected cells were immunolabeled using an anti-FLAG antibody (IF FLAG), and PLA was performed using anti-FLAG and anti–β-catenin (PLA β-cat/FLAG), anti–γ-catenin (PLA γ-cat/FLAG), or anti-PrP^c antibodies (PLA PrP^c/FLAG). Nuclei were stained by DAPI (bar, 20 μm). Graphs present the quantifications of PLA signal for each interaction normalized to values of IF FLAG signal, measured in at least 10 random fields (∼1000 cells/experiment) in two independent experiments (A.U., arbitrary units; mean ± SEM; ***p < 0.001 vs. PLA β-cat/FLAG). NLS, nuclear localization signal; DBD, DNA-binding domain. (B) Schematic diagram of the different FLAG-TCF7L2 deletion constructs. TCF7L2 lacking the β-catenin interaction domain (TCF7L2^{del 1-51}), lacking both β- and γ-catenin interaction domains (TCF7L2^{del 2-100}), lacking the 52–82 γ-catenin interaction domain (TCF7L2^{del 52-143}), or deleted for a sequence adjacent to the γ-catenin interaction domain (TCF7L2^{del 82-143}). Experiments were conducted as in A. Graphs present the quantifications of PLA signal normalized to values of IF FLAG signal for each construct. Measurements were performed as described in A. For each interaction, results obtained with the different constructs are presented as percentage of the value obtained with the full-length (FLAG-TCF7L2^FL) construct (mean ± SEM; **p < 0.01 and ***p < 0.001 vs. FL).