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. 2018 Nov 14;7(22):e010690. doi: 10.1161/JAHA.118.010690

Figure 14.

Figure 14

ADTRP negatively regulates Wnt/β‐catenin pathway. A, Western blot with anti‐FLAG and anti‐GAPDH IgGs in total lysates of HEK293T cells transfected with control or ADTRP‐FLAG plasmids. Shown: representative blot from 3 experiments. B, mRNA expression of LEF1 and AXIN2 in control and ADTRP‐expressing HEK293T cells. Gray bars: treatment with Wnt3a conditioned medium for 24 hours. Values (mean±SEM by 1‐way ANOVA with Bonferroni's multicomparison test) are fold‐change from control (arbitrarily set to 1.0) after correction for β‐2 MICROGLOBULIN as internal control. *P<0.05; **P<0.01. C, TOPFlash luciferase reporter activity and statistics (unpaired t test) in HEK293T cells expressing empty vector (control) or ADTRP‐FLAG, incubated with Wnt3a medium for 24 hours (gray bars). Data are mean±SEM. ****P<0.0001. D, Total lysates of HEK293T cells analyzed by SDS‐PAGE and immunoblotted with anti‐active β‐Catenin and anti‐GAPDH IgGs. E, Immunofluorescence and confocal microscopy with anti‐active β‐Catenin (Cy3, red) IgG and scatter plot and statistics of mean fluorescence intensity (MFI) expressed as mean arbitrary values (AU)±SD (unpaired t test) measured within nuclei (n cells/condition analyzed in at least 5 images recorded from 3 different experiments). White arrow: nuclear active β‐catenin. Blue, nuclei. Bars, 10 μm. ****P<0.0001. F, TOPFlash luciferase reporter activity and statistics in HEK293T cells incubated with 10 mmol/L of LiCl for 24 hours. Data are mean±SEM by unpaired t test. NS, P>0.05. G and H, TOPFlash luciferase reporter activity and statistics (1‐way ANOVA with Bonferroni's multicomparison test) in control and ADTRP‐expressing HEK293T cells at 48 hours after transfection with either full‐length β‐CATENIN or active β‐CATENIN–expressing plasmids (G, gray bars); or (H) constitutively active LRP6ΔN plasmid (gray bars). Data are mean±SEM. NS, P>0.05; ****P<0.0001. For all panels: Markers represent different experiments. ADTRP indicates androgen‐dependent tissue factor pathway inhibitor regulating protein; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; IgG, immunoglobulin G; LRP6, low‐density lipoprotein receptor‐related protein 6; LEF1, lymphoid enhancer binding factor 1; NS, not significant.