Figure 3: Characterization of FGFR1 KD variants. | Scientific Reports

Figure 3: Characterization of FGFR1 KD variants.

From: Conformational transition of FGFR kinase activation revealed by site-specific unnatural amino acid reporter and single molecule FRET

Figure 3

Kinase activity of FGFR1K3Y.TC (control) and H589BCNK.TC and L662BCNK.TC variants was compared. Time-course of auto-phosphoryaltion was performed using a bioluminescent ADP-Glo kinase assay (left panel). Samples of H589BCNK.TC and L662BCNK.TC before (−ATP) and after 45 min incubation with ATP (+ATP) were subjected to SDS-PAGE and Western blotting using anti-pY653/654 antibody; subsequent staining was with Amido Black (inset). Km values for ATP for FGFR1 KD variants (right panel) were calculated from data obtained in a kinase assay at increasing concentrations of ATP (0–500 μM) using poly(E4Y1) peptide as a substrate. See also Supplementary Fig. S2b.

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