Fluorescent Probes for α4β1 and Fn3 Integrin Binding

FITC-labeled α4β1-specific probe, 4-((N′-2-methylphenyl)ureido)-phenylacetyl-L-leucyl-L-aspartyl-L-valyl-L-prolyl-L-alanyl-L-alanyl-L-lysine (FITC-LDVP) and its unlabeled version (LDVP), were from Tocris Bioscience (Avonmouth, Bristol, UK). Human VCAM D1D2 (mature residues F1 to T202) were expressed and purified from HEK 293S GnTI^−/− cell line supernatants by affinity chromatography and gel filtration (Yu et al., 2013 (link)). VCAM D1D2 was fluorescently labeled with Alexa Fluor 488 NHS Ester (Thermo Fisher Scientific). Human Fn3_9-10 S1417C mutant (mature residues G1326 to T1509) and its synergy and RGD site (R1374A&P1376A&R1379A&S1417C&Δ1493-1496) mutated inactive version were expressed in Escherichia coli and purified as described (Li et al., 2017 (link); Takagi et al., 2001 (link)). Fn3_9-10 S1417C mutant was fluorescently labeled with Alexa Fluor 488 C5 maleimide (Thermo Fisher Scientific) at residue Cys-1417. Both Fn3_9-10 S1417C mutant and its inactive version were biotinylated with Maleimide-PEG11-Biotin at residue 1417 (Thermo Fisher Scientific) in PBS.

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Li J., Yan J, & Springer T.A. (2021). Low-affinity integrin states have faster ligand-binding kinetics than the high-affinity state. eLife, 10, e73359.

Publication 2021

Alexa Fluor 488 NHS Ester Alexa Fluor 488 C5 maleimide Maleimide-PEG11-Biotin

Affinity chromatography Alexa fluor 488 Alexa fluor 488 c5 maleimide Biotin Cell line Escherichia coli Ester Fitc Gel filtration Human Leucyl lysine Maleimide Vcam

Corresponding Organization :

Other organizations : Boston Children's Hospital, Harvard University

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Variable analysis

independent variables

FITC-labeled α4β1-specific probe, 4-((N′-2-methylphenyl)ureido)-phenylacetyl-L-leucyl-L-aspartyl-L-valyl-L-prolyl-L-alanyl-L-alanyl-L-lysine (FITC-LDVP)
Unlabeled 4-((N′-2-methylphenyl)ureido)-phenylacetyl-L-leucyl-L-aspartyl-L-valyl-L-prolyl-L-alanyl-L-alanyl-L-lysine (LDVP)
Human Fn3_9-10 S1417C mutant (mature residues G1326 to T1509)
Human Fn3_9-10 S1417C mutant with synergy and RGD site (R1374A&P1376A&R1379A&S1417C&Δ1493-1496) mutations

dependent variables

Binding of FITC-LDVP and LDVP to cells
Fluorescence intensity of Alexa Fluor 488-labeled VCAM D1D2 and Fn3_9-10 S1417C mutant

control variables

Human VCAM D1D2 (mature residues F1 to T202) expressed and purified from HEK 293S GnTI-/- cell line supernatants
Fn3_9-10 S1417C mutant and its inactive version expressed in Escherichia coli and purified

positive controls

None specified

negative controls

Fn3_9-10 S1417C mutant with synergy and RGD site (R1374A&P1376A&R1379A&S1417C&Δ1493-1496) mutations

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