Journal: Nature Communications
Article Title: Novel peptide probes to assess the tensional state of fibronectin fibers in cancer
Figure Lengend Snippet: In vitro binding studies of the peptide FnBPA5-Alexa-488 and the scrambled control (scraFnBPA5-Alexa-488) to soluble and fibrillar Fn. a Mechanosensitive binding of FnBPA5 was tested using relaxed (7% strain) and stretched (380% strain) fibers. Color-coded intensity ratio images of FnBPA5-Alexa488 divided by Fn-Cy5 of manually pulled Fn fibers of different mechanical state show higher binding to relaxed fibers than to stretched ones, leading to a higher FnBPA5/Fn ratio (representative images). Scale bar, 50 μm. b Normalized analysis of relaxed and stretched fibers showing a significant decrease in binding ratio of FnBPA5 from relaxed to stretched fibers. Data from 30 fibers from three independent experiments with error bars being standard deviations. p -value was obtained from a Student’s t test. c Schematic of manual fiber pulling, using a sharp pipette tip dipping into a Fn solution and pulling Fn fibers onto a flexible silicone sheet. d The binding of FnBPA5-Alexa-488 and scraFnBPA5-Alexa488 to soluble plasma Fn was determined using an anisotropy measurement (Methods). The results show a K d of 75 ± 8 nM for FnBPA5-Alexa488 and no specific binding for the scrambled control peptide. e Measurements of affinity of FnBPA5-Alexa488 to single relaxed Fn fibers showed a K d of 28 ± 6 nM. f Fluorescence images showing the binding of FnBPA5-Alexa488 and scraFnBPA5-Alexa488 to single relaxed Fn fibers (7% strain) using a previously described stretch assay 41 . For this experiment Cy5-labeled Fn was used (images on the left side). Scale bar, 50 μm. g The quantification of the fluorescence intensity showed a significant higher binding of FnBPA5-Alexa488 compared to the control. Data from 30 fibers from three independent experiments were analyzed. Shown error bars represent the standard deviation. p -value was obtained from a Student’s t test. h Human dermal fibroblast ECM was stained for Fn and incubated with FnBPA5-Alexa488 and the scrambled control peptide, respectively. Representative images show specific binding of FnBPA5-Alexa488 to Fn. Scale bar, 10 μm
Article Snippet: Primary antibody solution was removed and samples were washed before incubation with secondary goat anti-rabbit Alexa-633 (A-21070, Invitrogen, 1:100 dilution) or goat anti rat Alexa 546 (A-11081, Invitrogen, 1:100 dilution) or goat anti-rabbit Alexa-488 (A-11034, Invitrogen, 1:100 dilution) antibody solution for 45 min. After another washing step, samples were mounted using Prolong Antifade Gold with or without DAPI (Invitrogen).
Techniques: In Vitro, Binding Assay, Transferring, Fluorescence, Labeling, Standard Deviation, Staining, Incubation