Recombinant human loxl2

Extracellular LOX activity was quantified using a fluorometric activity assay kit (Abcam Cat. #112139) as previously described.^{55 (link)} Prior to activity assay, cells were switched to DMEM with no phenol red supplemented with 4 mM L-Glutamine, 10% FBS, and 1x pen/strep for 24 h. To measure extracellular LOX activity, conditioned medium was harvested directly from the plate. Activity assays were performed using 50 µl of conditioned medium mixed with 50 µl of reaction mix in a black-walled 96 well plate. Reactions were incubated for 30 min at 37°C protected from light and then the fluorescent signal (Ex540nm/Em590nm) was quantified on a BioTek Gen5 plate reader (Ex540 nm/Em590 nm). In all cases, signal from a medium-only control well was subtracted as the blank and recombinant human LOXL2 (R&D Systems Cat. # 2639-AO-010) was used as a positive control. To inhibit LOX, βAPN resuspended in water was added to growth or differentiation medium at a final concentration of 1, 2, 5, or 10 mM for the duration of differentiation. LOX rescue experiments were performed using 1 µg/ml recombinant human LOX (Origene Technologies Cat. #TP313323) or recombinant human LOXL2 added directly to differentiation medium for 4 d.

The binding of LOXL2 to α5β1 integrin was identified by Bio-Layer Interferometry, a label-free technique monitoring biomolecular interactions in real time, using an Octet RED96 system (FortéBio, Sartorius, Dourdan, France), as previously described [33 (link),72 ]. Recombinant human LOXL2 (R&D Systems, BioTechne, Noyal Châtillon sur Seiche, France, 2639-AO) was covalently immobilized via its primary amine groups on AR2G biosensors (FortéBio, Sartorius) at 25 ug/mL in sodium acetate buffer 10 mM pH 4. The sensors coated with LOXL2 and control sensors were dipped in human α5β1 integrin (Millipore, Merck, Molsheim, France, CC1027) diluted in 10 mM Hepes pH 7.4 containing 150 mM NaCl, 50 mM β-octyl-D-glucopyranoside, 1 mM MgCl₂, and 1 mM MnCl₂. Inhibition experiments were performed by preincubating the α5β1 integrin at 35.5 µg/mL for one hour at room temperature with or without the RGD peptide diluted at 50 µg/mL in the above buffer (Sigma–Aldrich, Saint-Quentin-Fallavier, France, A8052). Kinetics and affinity parameters were calculated using the evaluation software (version 9.0).