Salamander-Derived, Human-Optimized nAG Protein Suppresses Collagen Synthesis and Increases Collagen Degradation in Primary Human Fibroblasts
Figure 7
Determination of pro-MMP-2 and active MMP-2 by gelatin zymography ((a) and (b)) and pro-MMP-1 by ELISA (c) in nontransfected and nAG transfected fibroblasts. (a) Gelatin zymography: media were harvested after two days of transfection of nAG plasmid into fibroblasts and non-transfected fibroblasts (both were treated with 3 ng/mL TGF-β1). The first three lanes were different fractions of the same sample of non-transfected fibroblasts, the fourth lane was MMP-2 (positive control), and the last three lanes were samples of nAG transfected fibroblasts. (b) Densitometry analysis for gelatin zymography: density of each band was measured by using densitometry tool in Gel documentation software. There was 37% increase in pro-MMP-2 () and 85% increase in active MMP-2 () in transfected fibroblasts compared to non-transfected fibroblasts. (c) ELISA assay for pro-MMP-1 measurement: after two days of transfection. Media were harvested for measurement of pro-MMP-1 in non-transfected fibroblasts (control), nAG transfected fibroblasts, and fibroblasts treated with recombinant nAG. The level of pro-MMP-1 was 53-fold increase in transfected fibroblast compared to non-transfected fibroblasts () and it was 4-fold increase in nAG-treated fibroblasts compared to non-transfected fibroblasts ().
