Insight evaluation software

The SPR analyses were carried out using the Biacore 8K surface plasmon resonance instrument (Cytiva, Marlborough, MA, USA). A total of 100 nM His₆-14-3-3ε was immobilized on the NiCl₂-activated NTA chip (Cytiva, Marlborough, MA, USA) as previously described [34 (link)]. Then, different concentrations of peptide in the range of 0−500 μM (depending on the peptide) were injected. The experiments were performed in 20 mM TRIS, 150 mM NaCl, 50 μM EDTA, 0.005% Tween 20, and pH 7.4 buffer. The affinities of the peptides binding to 14-3-3ε were determined by fitting the data to a steady-state binding model using Biacore Insight Evaluation Software (version 5.0.18.22102).

Surface plasmon resonance (SPR) analysis by Biacore 8 K (Cytiva, Marlborough, MA) was performed at 25 °C in a run buffer of HBS-EP (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% v/v Surfactant P20, Cytiva) and with 15 mM HCl as regeneration solution. Recombinant human ErbB3/Her3 Fc (R&D Systems, Minneapolis, MN) was immobilized by standard amine coupling (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS)) on a CM5 sensor chip (Cytiva, Marlborough, MA) at ~ 1000 RU. The coated chip was preconditioned by three regeneration rounds to stabilize surfaces prior to injection of analyte. Binding of Affibody molecules to HER3 was analyzed by single cycle kinetics via injection of analyte at five different concentrations of purified Affibody molecule (1.25, 2.5, 5, 10 and 20 nM) over immobilized HER3/Fc. The experiment was performed in duplicates. Biacore Insight Evaluation Software was used to process, analyze and fit data.

In vitro interactions between Mps1, etc., and A378-0 were analyzed by surface plasmon resonance (SPR). The experiment was performed with the Biacore 8k+ instrument (Cytiva) at 25°C. Compound A378-0 was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 mg mL⁻¹ (17.7 mM). The running buffer for protein immobilization contained 1× PBS and 0.005% Tween 20, and that for compound analysis contained 1× PBS, 0.005% Tween 20, and 5% DMSO. The flow rates were 10 μL/min for protein immobilization and 30 μL/min for compound analysis. Proteins were immobilized on flow cell 2 of different channels of a series S CM5 sensor chip via an amine-coupling method to 5,000 to 6,000 response units (RU). Flow cell 1 of the corresponding channels of the sensor chip was left blank as a control. Compound A378-0 was injected with a concentration series of 6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM, and 200 μM, with one intermediate concentration injected as a duplicate. Both the association and dissociation periods were set to 60 s, and no regeneration was needed. Data were analyzed with Biacore Insight Evaluation software, and values from the first 5 s after the start of the injection were used to calculate the dissociation equilibrium constant (K_D) by fitting to a 1:1 steady-state affinity model.