Fig 1: Functional activity of digested 38B8 mAb in the TrkB NFAT reporter gene assays.CellSensor® TrkB-NFAT-bla CHO-K1 cells were stimulated with (A) BDNF, mAb 38B8, 38B8 Fab or 38B8 F(ab’)2 over the indicated concentration range for 5 hours (agonist mode) or (B) 38B8 Fab for 1 hour prior to 0.3 nM BDNF stimulation for 4 hours (antagonist mode) before beta-lactamase assay was performed as described in Methods. % of control (maximal BDNF concentration = 9 nM) values were plotted for the indicated concentrations of each ligand (n = 2 ± SD for each data point).
Fig 2: mHTT-induced neuronal toxicity: 7,8-dihydroxyflavone and LM22A-4 pharmacology.Using our primary rat cortico-striatal co-culture system we stimulated with 7,8-dihydroxyflavone, LM22A-4 or BDNF over the indicated concentration range according to the mHTT-induced co-culture protocol (A), as described in Methods. % Rescue (Normalized to in-plate controls; 1 nM BDNF (100% Rescue) and vehicle (0% Rescue)) values were plotted for striatal neurons over the indicated concentrations of each ligand (n = 6 ± SD for each data point). Rat primary cortico-striatal cells (non-transfected) were stimulated with 7,8-dihydroxyflavone (B) or LM22A-4 (C) using the indicated concentrations for 15 minutes and profiled by western blot. BDNF- (10 nM) mediated TrkB phosphorylation validated the experimental system.
Fig 3: Functional activity of TrkB mAbs in the primary cortico-striatal neuronal co-culture system.Rat primary cortico-striatal co-cultures were stimulated with BDNF, mAb 38B8 and mAb 29D7 and profiled by (A) western blot; agonists tested at 10 nM over the indicated incubation times using non-transfected co-cultures; or (B) mHTT-induced co-culture protocol over the indicated concentration range, as described in the Materials and Methods. For western blot, lysates were prepared and run on SDS-PAGE gels and transferred to membranes that were then hybridized to the corresponding primary antibodies, as described in the Methods and Materials. For mHTT-induced co-culture assay, % Rescue (Normalized to in-plate controls; 0.22 nM BDNF (100% Rescue) and vehicle (0% Rescue)) values were plotted for striatal neurons over the indicated concentrations of each ligand (n = 6 ± SD for each data point). Data demonstrates activation of TrkB phosphorylation signal transduction cascade and rescue of mHTT-induced neuronal toxicity by TrkB mAbs.
Fig 4: Literature-reported TrkB small molecule agonists.Literature-reporteded TrkB small molecule agonists tested were amitriptyline [33], 7,8-dihydroxyflavone [36], [37], N-acetyl serotonin [35], LM22A-4 [38], and BAG (cyclic peptide [39]. Compound structure or sequence as well as corresponding reported properties and activities are indicated.
Fig 5: TrkB signaling and assay cascades.(A) TrkB signalling cascade showing proximal and distal assay measurement points. Red arrows represent BDNF-induced trans-phosphorylation events within the intracellular tyrosine kinase domains. Changes in TrkB phosphorylation/activation detected by (1) Invitrogen CellSensor®; (2) DiscoveRx PathHunter®; and (3) MSD® pAKT assays. (B) Screening cascade used to characterize TrkB modulators.
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