Journal: bioRxiv
Article Title: Designed NGF mimetics with reduced nociceptive signatures in neurons
doi: 10.1101/2025.04.14.648806
Figure Lengend Snippet: Dose-response curves for pERK (middle) and pAKT (right) activation measured via phosphoflow for different backbone classes of all designs (colored) (A) Large designs (B) DHR04-based (C) 6-helical bundles (D) 6-helical short connector bundles (E) direct fusions and (F) direct long fusions. Error bars represent SD from three independent biological repeats. (G) Activation strength for the individual designs correlate for ERK and AKT phosphorylation. (H) Designs Nt-231 (top) and Nt-237 (bottom) were tested for specific TrkA pathway activation using a SRE-luciferase reporter gene and treatment with different concentrations of agonists. Designs did not lead to pathway activation of TrkB or TrkC. Error bars represent SEM from five independent biological repeats. (I) Time-course of TrkA receptor endocytosis for various concentrations of Nt-231 (top), Nt-237 (middle) agonists or NGF (bottom) using a BRET assay. Error bars represent SEM from five independent biological repeats.
Article Snippet: After blocking the membrane in 5% milk, 0.1% Tween, 10 mM Tris at pH 7.6, 100 mM NaCl, primary antibodies TrkA (Cell Signaling, Cat. 30697 at 1:1000), ERK (Cell Signaling, Cat. 9107), pERK (Cell Signaling, Cat. 4695), HSC70 (Santa Cruz, Cat. sc-7298; at 1:10.000), and Beta-Actin (Sigma, Cat. A1978 at 1:10,000) were added.
Techniques: Activation Assay, Phospho-proteomics, Luciferase, Bioluminescence Resonance Energy Transfer