Journal: bioRxiv
Article Title: Neuropilin-1 is a co-receptor for NGF and TrkA-evoked pain
doi: 10.1101/2023.12.06.570398
Figure Lengend Snippet: ( A ) Carboxy-terminal sequences of NGF, highlighting prospective CendR motifs (R/KxxR/K). Amino acids numbered according to the mature βNGF sequence (1-120 equivalent to proNGF 122-241). h, Homo sapiens; r, Rattus norvegicus; m, Mus musculus . ( B ) Prospective binding site between human βNGF and NRP1 modelled using protein docking PIPER software. ( C ) Interaction between purified His-tagged human NRP1 (residues 22-644) and unlabeled human beta-NGF using MST. Data from 4 independent experiments. Mean±SEM. ( D-E ) Bioluminescence resonance energy transfer (BRET) to measure proximity (<10 nm) at full-length NRP1 in living HEK293T cells at 37°C. Supernatant was collected from cells secreting growth factor (VEGF 165 a or NGF) tagged with HiBiT, a small portion of nanoluciferase with high affinity. When reconstituted with recombinant LgBiT and luciferase substrate, this acts as a bioluminescent donor for SnapTag-NRP1 labeled with SNAPTag-Alexa Fluor® 488 (AF488). BRET was compared to negative control (HiBiT/LgBiT only lacking AF488). Cells were pre-incubated with vehicle or 10 nM unlabeled VEGF 165 a (30 min), followed by luminescent growth factor (15 min, 37°C). BRET with HiBiT-VEGF 165 a was measured between HiBiT-VEGF 165 a and either SnapTag-NRP1 (WT) or the known VEGF 165 a binding-dead mutant (Y297A), as well as HiBiT-tagged NGF ( E ). Data from 4 independent experiments with triplicate wells. F. 1-way ANOVA, Šídák’s multiple comparisons. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001.
Article Snippet: Recombinant βNGF (R&D Systems or Alomone Labs) and recombinant NRP1 (R&D Systems) were reconstituted according to manufacturer’s instructions.
Techniques: Sequencing, Binding Assay, Software, Purification, Bioluminescence Resonance Energy Transfer, Recombinant, Luciferase, Labeling, Negative Control, Incubation, Mutagenesis
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