Figure 6.

The NRP1 Mutant Y297A Is Unable to Bind Any VEGF Isoforms

(A) Live confocal imaging of HEK293T cells stably expressing mutant HaloTag-NRP1 Y297A (green) labeled with membrane-impermeant HaloTag-AF488 dye (green). Cells were stimulated with 10 nM VEGF₁₆₅a-TMR, VEGF₁₆₅b-TMR, or VEGF₁₂₁a-TMR for 60 min at 37°C. Cells were imaged using an LSM710 confocal microscope, and images are representative images of 3 independent experiments. Scale bar, 10 μm.

(B) NanoBRET measurements of the effect of unlabeled VEGF isoforms (30 nM) on the binding of 3 nM VEGF₁₆₅a-TMR to wild-type NanoLuc-NRP1 or NanoLuc-NRP1 Y297A stably expressing HEK293T for 60 min (37°C). Raw BRET ratios are expressed as mean ± SEM pooled from 4 independent experiments.