Biophysical definition of the Nrxn1β/FAM19A1 complex. (A and B) Nrxn1β-ECD (Nrxn1β-ECD-Myc-6xHis) was coexpressed with FAM19A1-V5 in HEK293S GnTI⁻ cells and purified from the medium. In the final purification step, the Nrxn1β-ECD/FAM19A1 complex elutes as a single peak (A, peak 1), as shown by Coomassie-blue staining (B) and immunoblotting for Myc and V5 (A, inset), whereas excess Nrxn1β-ECD elutes as a separate peak (A, peak 2). (C andD) Final purification step for Nrxn1β-ECD-Myc-6xHis, expressed alone, from the medium of HEK293S GnTI⁻ cells (C, elution profile; D, Coomassie-blue-stained reducing SDS-gel). (E and F) Final purification step for FAM19A1-Twin-Strep, expressed alone, from the medium of HEK293S GnTI⁻ cells (E, elution profile with Twin-Strep immunoblot in inset; F, Coomassie-blue-stained reducing SDS-gel). See also Fig. S3, H–P. (G and H) Nrxn1β-ECD forms a stoichiometric 1:1 complex with FAM19A1. Analysis by size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) of the purified Nrxn1β-ECD/FAM19A1 complex and of Nrxn1β-ECD (G), and of FAM19A1 (H), which enables the determination of their precise molar masses (dRI, differential refractive index; MM, molar mass; dotted lines in B, D, and F denote fractions that were combined and analyzed). (I) Molar masses of the Nrxn1β-ECD/FAM19A1 complex, Nrxn1β-ECD, and FAM19A1 as predicted (Prot pi Protein Tool; assuming all cysteines form disulfide bonds and Nrxn1β contains one Man₅GlcNAc₂ addition) or as experimentally determined by MALS or electrospray ionization (ESI)-MS.