Fig 2: Expression, purification, and biotinylation of recombinant NID2 proteins. (A) SEC profile of purified NID2‐FL‐Avi on a Superdex™ 200 Increase 10/300 GL column. The main peak eluted at approximately 9–11 mL. Fractions corresponding to the main peak were analyzed by SDS/PAGE. (B) SEC profile of biotinylated NID2‐FL‐Avi‐biotin on a Superdex™ 200 Increase 10/300 GL column. Biotinylation was confirmed by streptavidin–HRP blotting. (C) SEC purification of NID2‐FL on a Superdex™ 200 Increase 10/300 GL column and SDS/PAGE analysis of peak fractions. (D) SEC purification of NID2‐G1G2 on a Superdex™ 200 Increase 10/300 GL column. The main peak eluted at approximately 10.5 mL, and corresponding fractions were analyzed by SDS/PAGE.

Fig 3: Epitope competition analysis of anti‐NID2 nanobodies targeting the rod–G3 domain by BLI. (A–F) Representative BLI competition sensorgrams showing sequential binding of rod–G3–targeting nanobodies to immobilized NID2‐FL‐Avi. Based on the competition patterns observed in the second association step, the nanobodies were classified into five distinct, non‐overlapping epitope groups within the rod–G3 domain. Panels (A–F) show representative data from a single experiment (no biological replicates).

Fig 4: Domain organization of human NID2. The schematic illustration is based on protein structure prediction using alphafold3, with domains colored as G1 (purple), G2 (magenta), and G3 (cyan). The illustration highlights the flexible intrinsically disordered linker connecting the G1 and G2 domains and the rigid rod‐like region composed of EGF‐like repeats linking the G2 and G3 domains. Domain boundaries, residue ranges, and annotated post‐translational modification (PTM)–prone residues are indicated to facilitate interpretation of domain‐specific nanobody binding.

Fig 5: Affinity Determination Results of Anti‐NID2‐FL Nanobodies. (A) Representative binding and dissociation sensorgrams of NB‐2E6 interacting with immobilized NID2‐FL‐Avi at the indicated concentrations. (B) Representative binding and dissociation sensorgrams of NB‐1E5 interacting with immobilized NID2‐FL‐Avi at the indicated concentrations. (C) Representative binding and dissociation sensorgrams of NB‐1B5 interacting with immobilized NID2‐FL‐Avi at the indicated concentrations. (D) Representative binding and dissociation sensorgrams of NB‐1C3 interacting with immobilized NID2‐FL‐Avi at the indicated concentrations. For NB‐2E6 and NB‐1C3, dissociation rates reached the lower detection limit of the Octet system; therefore, the reported KD values represent apparent affinities constrained by instrumental sensitivity. Each concentration was measured once; panels show representative sensorgrams from these single measurements (no biological replicates).