Glycine 1

SPR measurements were performed using a Biacore ×100 SPR system (GE Healthcare). Human IL4 receptor alpha-FC chimera (Biolegend) was immobilized on a protein G sensor chip (GE Healthcare). Log₂ dilution concentration series consisted of apoA1–IL4 ranging from 200 nM to 6.25 nM and of human IL4 ranging from 20 nM to 0.65 nM. All samples were prepared in HPS-EP buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% (v/v) P20 pH 7.4). Association was monitored for 180 s and dissociation for 180 s with a flow rate of 30 μl min⁻¹. Sensor chip was regenerated with glycine 1.5 (10 mM glycine-HCl pH 1.5, GE Healthcare). Kinetics was determined by fitting the interaction SPR data for 1:1 binding.

Binding kinetics of the antibodies against human, cynomolgus monkey, or mouse latent myostatin were assessed at pH 7.4 and 6.0 at 25 °C using the Biacore T200 (GE Healthcare Life Sciences, Piscataway, NJ). Antibodies were captured onto the Biacore sensor chip CM5 (GE Healthcare Life Sciences) and immobilized with protein L (BioVision). Recombinant human, monkey, or mouse latent myostatin was prepared by two-fold serial dilutions (2 nmol/L to 32 nmol/L). The sensor surface was regenerated using Glycine 1.5 (10 mmol/L glycine–HCl, pH 1.5, GE Healthcare Life Sciences). Kinetic parameters at pH 7.4 were determined by fitting the sensorgrams to the 1:1 binding model using the Biacore T200 Evaluation Software, version 2.0 (GE Healthcare Life Sciences). The pH-dependent binding ability of the antibodies to latent myostatin was evaluated by comparing the dissociation phases of the sensorgrams at pH 7.4 and at pH 6.0. Further details are described in the Supplementary Materials.