Streptavidin coated sa chip

10 μg/ml calf bulk histone was dissolved in coating buffer (50 mM sodium acetate [pH 5.0]) and coated onto a CM5 chip (GE Healthcare). Different concentrations of JMJD7 protein were then injected to calculate the binding affinity between bulk histone and JMJD7. Peptides were ordered from AnaSpec Inc. The sequence for the peptides used in our experiment are listed in Table S7. For peptides, 2 μg/ml synthetic peptide with C-terminal biotin was dissolved in PBS and coated onto a streptavidin coated SA chip (GE Healthcare). Different concentrations of JMJD7 protein were then injected to calculate the binding affinity between peptide and JMJD7. Similar procedures were carried out for JMJD5. All experiments are carried out at room temperature.

Interaction experiments using surface plasmon resonance (SPR) were carried out with a Biacore T200 system (GE Healthcare) according to the manufacturer’s protocols and recommendations. Experiments were setup as described before^{23 (link)}. Briefly, purified biotinylated ACE2^{23 (link)} was immobilized on a streptavidin-coated (SA) chip (GE Healthcare) at ~50 RUs. BriSΔ was injected at concentrations of 40 nM, 80 nM,120 nM, and 160 nM. S^ΔRBM, S^RRAR->A, S^RRAR, and S^RRAR* were injected at 40 nM and 160 nM. The running buffer for all measurements was PBS buffer pH 7.5. Sensorgrams were analyzed and K_D, k_on and k_off values were determined with the Biacore Evaluation Software (GE Healthcare), fitting the raw data using a 1:1 binding model. All experiments were performed in triplicates.

KRAS^G12V, HRAS^wt and NRAS^wt (all amino acids 1–166) were expressed in E.coli C41 after cloning in the pRK-HIS-TEV-Avi vector, and purified as described using^{19 (link)}. The protein extracts were loaded with GPPNHP (a non-hydrolysable GTP analogue) as described^{19 (link)} and biotinylated by incubation with BirA overnight at 4 °C. Final purification was carried out by gel filtration through Superdex 75. SPR experiments were performed using BIAcore T100 (GE Healthcare). The biotin-KRAS[G12V]-GPPNHP, Biotin-HRAS-GPPNHP and Biotin-NRAS-GPPNHP were immobilised on a streptavidin-coated SA chip (GE Healthcare). The chip was prepared with 3 × 30 s injections of 1 M NaCl/50 mM NaOH at 10 µL/min flow rate, before biotinylated RAS proteins were injected at 25 µg/mL with 10 µL/min flow rate until 4000–5000 RU protein was captured. The immobilisation buffer consisted of 10 mM HEPES pH 8.0, 150 mM NaCl, 1 mM MgCl₂. Compound binding experiments were performed at 25 °C using multi-cycle injections of compound at 10 concentrations between 0.312–100 µM. The flow rate was 30 µL/min in running buffer consisting of 10 mM HEPES pH 8.0, 150 mM NaCl, 1 mM MgCl₂, 0.05% Surfactant P20, 5% DMSO. Data were reference subtracted and DMSO solvent correction applied, before being fitted to a Steady State Affinity 1:1 binding model using Biacore T200 Evaluation Software version 2.0.