Figure 3

Catalytically inactive HTRA1 affects Wnt signaling. (A) HEK293T cells were transfected with HTRA1-GFP, HTRA1 S328A-GFP, or an empty pEGFP vector as a basal control (2 μg). Forty-eight hours after transfection, CM was collected and the cells were harvested. Both the medium and the lysates were subjected to Western blot analysis, using anti-GFP or anti-tubulin antibodies. (B) HEK293T cells were transfected with the pTOPFLASH/pFOPFLASH constructs (0.5 μg) and β-gal plasmid (0.1 μg), along with HTRA1-GFP, HTRA1 S328A-GFP, GFP-β-catenin, or an empty pEGFP vector as a basal control (1 μg). Forty-eight hours after transfection, cells were harvested and subjected to a luciferase assay or Western blot analysis, using anti-HA, anti-HTRA1, or anti-tubulin antibodies. (C) HEK293T cells were transfected with HTRA1-GFP, HTRA1 S328A-GFP, or an empty pEGFP vector as a basal control (2 μg). Forty-eight hours after transfection, cells were incubated with 50 μM serine protease inhibitor 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) or dH₂O as a control for 30 minutes at 37 °C. Cells were harvested and total RNA was extracted and converted into a more stable cDNA. The cDNA was subjected to real-time PCR assay as described earlier. (*p value < 0.05 **p value < 0.01, Student’s t-test).