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Fig. 1 | Cell Communication and Signaling

Fig. 1

From: Protease associated domain of RNF43 is not necessary for the suppression of Wnt/β-catenin signaling in human cells

Fig. 1

Inducible RNF43 and RNF43ΔPA expression in T-REx 293 cell line. a. Schematic representation of the used experimental models. T-REx 293 cell lines inducibly expressing tagged wild type RNF43 or its variant lacking PA (aa 87–186) domain. Supplementation of culture medium with Tet drives transgene expression. b. Western blot showing Tet-induced expression of HA-tagged RNF43 constructs in stable sell lines. β-actin served as loading control. c. Expression of RNF43 (c), ZNRF3 (c’) and CTNNB1 (c”), genes in response to the 80 ng/ml rWNT3A, 50 ng/ml rRSPO1 and tetracycline overnight treatments in the parental, RNF43 and RNF43ΔPA (clone #1 and clone #2) TetON cell lines assessed by the qPCR. Data are presented as relative expression, 2−ΔΔCt ± SD. d. Immunofluorescence of RNF43 and RNF43ΔPA intracellular localization. GFP with plasma membrane targeting signal served as plasma membrane marker (in green), HA tag for the detection of exogenous RNF43 and RNF43ΔPA mutant (in red). Nuclei stained with TO-PRO-3 Iodide are shown in blue. Scale bars represent 25 μm. Signals intensities were measured along selections and plotted. e. T-REx 293 cells overexpressing RNF43 were compared with the parental cell line for their ability to mediate TCF/LEF-dependent transcription in the Top flash dual luciferase assay. Response to 80 ng/ml rWNT3A, 25 ng/ml rRSPO1 and Tet treatments was measured after overnight incubation. Values are normalized to the unstimulated control cells. N = 4, unpaired two-tailed t-test **p < 0.01, ***p < 0.001. f. Western blot analysis of canonical Wnt pathway activation by application of the increasing concentrations of rWNT3A (40, 60 and 100 ng/ml) after 3 h of treatment. In cells overexpressing RNF43 phosphorylation of S1490-LRP6 and DVL2 (arrowheads) were weaker. Signal corresponding to the β-actin signal was used as loading control and HA tag specific antibody for RNF43 detection, N = 3

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