Figure 3.

Loss of RhoGEF17 results in the accumulation of phosphorylated β-catenin and increases β-catenin target gene expression in EC. (A) HUVEC or (B) RFPEC were transduced for 48 h. Representative immunoblots of β-catenin and α-tubulin are shown. The intensity of both detected β-catenin bands (β-cat) as well as the intensity of the upper β-catenin band (mod-β-cat) were quantified and normalized by α-tubulin. The values are given relative to the EGFP control as means + SEM with the single data points, n = 3 (HUVEC), n = 3–14 (RFPEC), * p < 0.05 vs. EGFP control (not shown) assessed by paired t-testing. (C) The proteasome was inhibited with 100 nM Bortezomib for 2 h in RFPEC. β-catenin and phosphorylated β-catenin were detected by immunoblot. Shown are representative immunoblots of β-catenin (Ab1 = Santa Cruz, Ab2 = Zymo Research), p-β-catenin (S33/37/T41) and α-tubulin (left) and the quantitative analyses. The values are normalized and given as means + SEM with the single data points, n = 8–11, * p < 0.05 assessed by 2-way ANOVA with Tukey’s multiple comparison test. (D) Axin1, survivin, cyclin D1 and α-tubulin were detected by immunoblot in RFPEC lysates. Shown are representative immunoblots (left) and the corresponding analyses. The values are given relative to the EGFP control as means + SEM with the single data points, n = 3, * p < 0.05 vs. EGFP control (not shown) assessed by paired t-testing. (E) Cell fractionation experiments were performed with transduced RFPEC. β-catenin, histone H1 and GAPDH were detected by immunoblot in different cell fractions.