Figure 4

HTRA1 interacts with β-catenin. (A) HEK293T cells were transfected with the pTOPFLASH/pFOPFLASH constructs (0.5 μg) and β-gal plasmid (0.1 μg), along with HTRA1-HA, HA-β-catenin, or an empty HA vector as a basal control (1 μg). Forty-eight hours after transfection, cells were treated for five hours with either MG132 (25 µM) or DMSO as a control. The cells were harvested and subjected to a luciferase assay. (*p value < 0.05 **p value < 0.01, Student’s t-test). (B) SW480 and HCT116 cells were transfected with HTRA1-GFP (1 μg). Forty-eight hours after transfection, cells were fixed, permeabilized, and stained with an anti-active β-catenin (ABC) and anti-golgin97 antibodies. Alexa-546 and Alexa-647 (respectively) were used as fluorescent antibodies and HTRA1 was detected through the GFP tag. Cells were visualized using confocal microscopy. (C) SW480 or HCT116 cell lysates were subjected to co-immunoprecipitation using an anti-β-catenin antibody or a non-specific IgG control. Lysates and immunoprecipitants were analyzed by western blotting using anti-HTRA1 or anti-β-catenin antibodies. (D) SW480 and HCT116 cells were immunostained with the rabbit anti-HTRA1 (ABGENT) and the mouse anti-total-β-catenin (BD) specific antibodies, as described. Alexa-546 and Alexa-488 (respectively) were used as fluorescent secondary antibodies.