fluorogenic peptide substrate  (R&D Systems)

Journal: bioRxiv

Article Title: Discovery, characterisation and optimisation of bicyclic peptide inhibitors that disarm Staphylococcus aureus α-hemolysin

doi: 10.64898/2026.03.09.710508

Figure Lengend Snippet: a) Alexa Fluor 647-labeled AhlyH35A (7.5nM) was incubated with A549 cells in the presence of increasing concentrations of Peptide 88 or a control bicyclic peptide. Cell-associated fluorescence was quantified by flow cytometry and shown as histogram overlays. Negative control (cells only) shown in black; positive control (AhlyH35A without peptide) shown in red. b) Quantification of median fluorescence intensity plotted against peptide concentration. Data are normalized to the negative and positive controls. c) ADAM10 protease activation by Ahly (6µM) was measured using a whole-cell FRET peptide cleavage assay in the presence of Peptide 88 or a control bicyclic peptide (900µM). Mean of two biological replicates; error bars indicate standard deviation. Data were analysed using one-way ANOVA with Dunnett’s test: ns = not significant; ** = P < 0.01.

Article Snippet: Following incubation, cells were washed once with 25mM Tris buffer, pH 8.0 and a fluorogenic ADAM10 substrate peptide (Mca-PLAQAV-Dpa-RSSSR-NH 2 ; R&D Systems) was added at a final concentration of 10μM.

Techniques: Labeling, Incubation, Control, Fluorescence, Flow Cytometry, Negative Control, Positive Control, Concentration Assay, Activation Assay, Cleavage Assay, Standard Deviation

Journal: iScience

Article Title: 2′-Fucosyllactose transactivates EGF receptor in intestinal epithelial cells for prevention of colitis in adulthood

doi: 10.1016/j.isci.2025.114308

Figure Lengend Snippet: 2′-FL transactivates EGFR in IECs via stimulating the release of HB-EGF from the cell membrane to bind to EGFR (A and C) Cells were treated with 2′-FL at indicated concentrations or 20 μg/mL of lactose or glucose for 2 h (A), or 2′-FL at 20 μg/mL for 2 h with and without 1-h pretreatment of HB-EGF neutralizing antibody at 500 ng/mL that was present with 2′-FL treatment (C). Total cellular lysates were prepared for Western blot analysis of the phosphorylated and total levels of EGFR and ERK1/2. β-actin blot was used as the protein loading control. (B) Cells were treated with 2′-FL at 20 μg/mL for indicated times. Cell culture supernatants were collected for ELISA analysis of HB-EGF levels. Data are presented as pg of HB-EGF in culture supernatant from 105 cells. (D) YAMC cells were treated with 2′-FL, lactose, or glucose at 20 μg/mL for 2 h. At the end of the treatment, the fluorogenic peptide substrate (4 μM) was added to cell culture medium and incubated for 2 h. The fluorescent intensity in supernatants was measured at 30, 60, 90, and 120 min after reaction. Data are presented as relative fluorescence unit (RFU)/minute in the indicated reaction period from 104 cells. Images in (A) and (C) represent data from at least three independent experiments. In (B) and (D), data were triplicated in each experiment, and the symbol represents the average data from individual experiment. Statistical significance was determined using unpaired t -tests and one-way ANOVA analysis of variance. ∗ p < 0.05 compared to the control group of the same cell line in (B). ∗ p < 0.05 compared to the control, glucose, and lactose groups in the same reaction period in (D). Data are represented as mean ± SD.

Article Snippet: 0.6 mL of RPMI medium/well containing 4 μM fluorogenic peptide Substrate III (Mca-P-L-A-Q-A-V-Dpa-R-S-S-S-R-NH2, #ES003, R&D Systems) was added to cell cultures.

Techniques: Membrane, Western Blot, Control, Cell Culture, Enzyme-linked Immunosorbent Assay, Incubation, Fluorescence