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. 2021 Feb 4;22(4):1575. doi: 10.3390/ijms22041575

Figure 7.

Figure 7

Fibrotic matrix binding characterization of the designed blocking polypeptide. The designed AF38Pep blocking polypeptide was synthesized and conjugated to Cy5 (AF38Pep-Cy5). (A) The extracellular matrices (ECMs) of fibroblasts or differentiated myofibroblasts were fixed and co-labelled with fibronectin (FN1) antibody and AF38Pep-Cy5. Original magnification, ×200; scale bar, 50 µm. (B) Binding competitiveness was assessed by preincubating fixed matrices with either AF38Pep-Cy5 or IST-9 antibody, followed by incubation with IST-9 or AF38Pep-Cy5, respectively. Original magnification, x200; scale bar, 50 µm. (C) Top panel (fibrotic matrix specificity): TGF-β1 was used to stimulate EDA-FN production at the indicated timepoints and 10 µg/mL unlabeled AF38Pep or AF38Pep-Cy5 was added for 1 h before washing and visualization of epifluorescence. Bottom panel (binding specificity): TGF-β1 was used to stimulate EDA-FN production at the indicated timepoints and 10 µg/mL IST-9 antibody was preincubated with cells for 1 h. Cells were washed and then 10 µg/mL unlabeled AF38Pep or AF38Pep-Cy5 was added for 1 h before washing and visualization of epifluorescence. Data are representative of three independent experiments and displayed as the mean ± S.E. Statistical analysis is shown as * p ≤ 0.05, and ns = no statistical significance (p > 0.05).