Figure 5

The effect of HTRA1 on Wnt signaling is mediated via its kazal and PDZ domains. (A) HEK293T cells were transfected with GFP-IGFBD, GFP-Kazal, GFP-protease, GFP-PDZ, or an empty pEGFP vector as a control (2 μg). Forty-eight hours after transfection, cells were harvested; total RNA was extracted from the cells and converted into a more stable cDNA. Quantitative real-time PCR was performed as described (*p value < 0.05 **p value < 0.01, Student’s t-test). (B) SW480 cells were co-transfected with HTRA1-GFP, GFP-IGFBD, GFP-Kazal, GFP-protease, GFP-PDZ, or an empty pEGFP vector as a control (1 μg) along with the pTOPFLASH/pFOPFLASH constructs (0.5 μg) and renilla plasmid (0.1 μg). Forty-eight hours after transfection, cells were harvested and subjected to a luciferase assay as described previously. Right panel depicts western blot analysis of the different HTRA1 domains (detected by anti-GFP antibody). (C) SW480 cells were co-transfected with two concentrations of the GFP-protease domain or an empty pEGFP vector as a control, along with the pTOPFLASH/pFOPFLASH constructs (0.5 μg) and renilla plasmid (0.1 μg). Forty-eight hours after transfection, cells were harvested and subjected to a luciferase assay as described previously. Upper panel depicts western blot analysis showing that the higher DNA concentration lead to an increased protein expression level (detected by anti-GFP antibody).