Fluorescamine assay

RGD-containing peptide sequence (GGGGRGDSP, GeneScript) was modified with an azide group via the terminal amine group. Briefly, RGD was dissolved in sodium bicarbonate buffer (pH 8.3) and NHS–C3-azide in DMSO was added in equimolar amount. The mixture reacted for 4 ​h at RT with agitation. Azide incorporation was indirectly monitored by quantifying the consumption of free amines with the fluorescamine assay (Sigma-Aldrich). Fluorescence readings (λ_ex/em: 400/460 ​nm) before and after reaction were compared with a glycine standard curve (Fig. S1), and the reaction efficiency was determined to be 81.07%.

Tissue resections were weighed and ground while frozen, and 30–50 mg aliquots were stored at −80°C. For extraction, a volume of 100 μL of buffer A (45 mM HEPES, 0.4 M KCl, 1 mM EDTA, 0.1 mM DTT, 10% glycerol, pH 7.8) was added to every 50 mg of ground tissue. Samples were vortexed, snap frozen, and 30 μL of 1% Triton X-100 in buffer A added per 100 μL. Protein concentration was measured by a fluorescamine assay (Sigma-Aldrich), on a NanoDrop 3300 (Thermo Scientific). Undiluted extracts were kept at −80°C. Before the incubation reaction, on the day of use, extracts were diluted to a protein concentration of 3 mg/mL in buffer A in a final volume of 50 μL and mixed with 4 volumes of buffer B.