Fig. 4: The C-terminal tail of AQP1 and GSK3β competitively interacted with the 12 armadillo repeats of β-catenin, then inhibited β-catenin degradation.

a Western blot analyses of β-catenin and active β-catenin in vector/MDA-MB-231 and AQP1/MDA-MB-231 cells, β-actin was the loading control. b RT-qPCR results of mRNA level of β-catenin in vector/MDA-MB-231 and AQP1/MDA-MB-231 cells. GAPDH was used as control. Values were expressed as mean ± SEM (two-tailed Student’s t test, ***P < 0.001). c, d Vector/MDA-MB-231 and AQP1/MDA-MB-231 cells were treated with 1 μM MG132 (c) or 100 μg/ml CHX (d) and harvested at the noted time points followed by western blot analyses. Here the β-actin was the loading control. e Vector/MDA-MB-231 and AQP1/MDA-MB-231 cells were extracted and immunoprecipitated with β-catenin antibody and then immunoblotted with ubiquitin antibody, β-actin was the loading control. f Western blot of lysates of vector/MDA-MB-231 and AQP1/MDA-MB-231 cells were analyzed with indicated antibodies, β-actin was the loading control. g Co-immunoprecipitation with Flag antibody showed the interaction of Flag-AQP1-ΔCT or Flag-AQP1-CT with β-catenin. h AQP1/MDA-MB-231 and AQP1-ΔCT/MDA-MB-231 cells were extracted and immunoprecipitated with β-catenin antibody and then immunoblotted with ubiquitin antibody, β-actin was the loading control. i Western blot of lysates of AQP1/MDA-MB-231 and AQP1-ΔCT/MDA-MB-231 cells were analyzed with indicated antibodies, β-actin was the loading control. j 12 armadillo repeats (12 × arm) of β-catenin mediated the interaction of β-catenin with AQP1 in co-immunoprecipitation experiments. k 12 armadillo repeats (12 × arm) of β-catenin mediated the interaction of β-catenin with GSK3β in co-immunoprecipitation experiments. l Endogenous GSK3β was immunoprecipitated. The interaction of GSK3β with β-catenin was analyzed by immunoblotting for β-catenin in MDA-MB-231 cells in the presence or absence of AQP1. Whole-cell lysates were directly subjected to western blot using Flag, GSK3β, and β-catenin antibodies as input. m Endogenous β-catenin was immunoprecipitated and interaction of GSK3β with β-catenin was analyzed by immunoblotting for GSK3β in Flag-AQP1/MDA-MB-231 and Flag-AQP1-ΔCT/MDA-MB-231 cells. n Cell viability of AQP1/MDA-MB-231 and AQP1-ΔCT/MDA-MB-231 with different concentration of EPI treatment for 48 h was tested in MTT assay. Values were expressed as mean ± SEM (two-tailed Student’s t test, **P < 0.01). All Experiments were independently repeated for three times.