Br100399

Surface plasmon resonance analysis was performed with Biacore X100 system (GE Healthcare). Recombinant CRM1 and p53 protein were immobilized on an activated CM5 dextran chip (GE Healthcare, BR100399), respectively. The binding of TPR at different concentrations in the presence or absence of TLNC1 was performed with PBS containing 500 mM NaCl (pH 7.4) and 0.1% Tween 20. The flow rate was set at 30 μL/min. The binding kinetics was analyzed by BIAevaluation software using the 1:1 L binding model.

Streptavidin was immobilized on the surface of a CM5 sensor chip (GE Healthcare, BR100399) using the amine coupling kit (GE Healthcare, BR100050). A solution containing EDC (7.5 mg mL⁻¹) and NHS (11.5 mg mL⁻¹) was injected into the flow cells to activate the carboxyl groups followed by a solution containing streptavidin (0.5 mg mL⁻¹) and finally ethanolamine (1 M) to block remaining activated carboxyl groups. All solutions were flowed through the channels for 7 min at a flow rate of 10 µL min⁻¹. Finally, biotinylated CA K158Cor biotinylated CA hexamer were immobilized on the surface by injection a 2.5 µM solution for 30 s at a flow rate of 30 µL min⁻¹. Flow channels modified with streptavidin were used as reference cells. CypA was buffer exchanged into SPR running buffer using a Zeba gel filtration spin column. CypA solutions at a range of concentrations (3.125–100 µM) were flowed through the cells (20 s at a flow rate of 100 µL min⁻¹) followed by a buffer wash (30 s at a flow rate of 100 µL min⁻¹) while measuring the SPR response at a frequency of 40 Hz.

Direct binding of IL-33 to the extracellular domain of ST2 was determined by surface plasmon resonance using a BIAcore 2000 (GE Healthcare). ST2 was immobilised using an anti-human Fc capture (GE Healthcare BR-1003-39) on a CM5 sensor chip (GE healthcare BR-1003-99) to give a stable surface of ∼150 RU. IL-33 was flowed over the surface at 30 μl min^–1 for 3 min to determine association rates. Dissociation was measured by flowing buffer at 30 μl min^–1 for 15 min. Sensorgrams were interpreted using BIAevaluation software and kinetics were determined using double reference subtracted sensorgrams using a 1:1 (Langmuir) binding model.