Fig. 4: Receptor activation via dye-labeled collagen-binding proteins.

a Schematic of OG488-CNA35 (CNA35 in blue, HaloTag in yellow, OG488 in green) bound to natively folded collagen-I (purple). b OG488-CNA35 activates αFITC(E2)-SynNotch specifically on collagen-I coated wells at low nanomolar concentration. HEK293-FT H2B-mCherry reporter cells were transiently transfected with plasmids encoding αFITC(E2)-SynNotch and mTurq2 as a cotransfection marker. Mean percent mCherry positive cells of the mTurq2+ population analyzed by flow cytometry from three independent transfections (n = 3 > 5000 cells per replicate) displayed +/- standard deviation. c Median mCherry fluorescence intensities showing clonal αFITC(E2)-SynNotch-mTurq2 cells specifically activated by 3 nM OG488-CNA35 bound to triple-helical collagen-I. Three independent OG488-CNA35 treatments (n = 3 > 5000 cells analyzed per replicate). Data presented as mean values +/- standard deviation and analyzed by one-way ANOVA, labeled NS: P > 0.05, ****: P < 0.0001. d Schematic of CHP-5-fluorescein bound to denatured collagen (gray). e CHP-5-fluorescein activates on heat-denatured collagen-I. HEK293-FT H2B-mCherry reporter cells were transiently transfected with αFITC(E2)-SynNotch and mTurq2 cotransfection marker. The mean percent mCherry positive cells of the mTurq2+ population analyzed by flow cytometry from three independent transfections (n = 3 > 5000 cells per replicate) displayed +/- standard deviation. f Median mCherry fluorescence intensities showing clonal αFITC(E2)-SynNotch-mTurq2 cells specifically activated by 2 µM CHP-5-fluorescein bound to heat-denatured collagen-I. Three independent CHP-5-fluorescein treatments (n = 3>5000 cells analyzed per replicate). Data presented as mean values +/- standard deviation and analyzed by one-way ANOVA, labeled NS: P > 0.05, ****: P < 0.0001. Data and P values, and non-linear sigmoidal curve fit are provided in the Source Data file. Components of the cartoons in (a) and (d) were created in BioRender⁸⁷.