Extended Data Fig. 2: SCG2 does not bind LILRB4 F83A mutant.
From: Secretogranin 2 binds LILRB4 resulting in immunosuppression
a, Mapping SCG2-binding domain via LILRB4-D1 truncation constructs. HEK293T cells were co-transfected with Flag-tagged LILRB4-D1 truncation mutants and full-length SCG2. After 48 hours, cell lysates were subjected to IP with anti-Flag beads, IB with indicated antibodies (same below). b, Identification of SCG2-binding region with AA55-123 of LILRB4. HEK293T cells were co-transfected with SCG2 and FLAG-tagged LILRB4 mutants harboring internal deletions within amino acids 55–123 (AA55–123). Interactions were assessed by IP and immunoblotting. c, Single amino acid mapping within AA75-84. HEK293T cells were co-transfected with SCG2 and Flag-tagged LILRB4 single-point mutants containing single amino acid substitutions within the AA75–84 region. Interactions with SCG2 were analyzed by Co-IP. d, Activation assays comparing LILRB4 wild-type and F83A mutant reporter cells stimulated with plate-coated recombinant SCG2 protein or coated anti-LILRB4 antibody as a positive control. Reporter cells were incubated for 24 hours at 37 °C, and GFP⁺ cells were quantified by flow cytometry (n = 3 biological replicates). Data are present as mean ± s.d. e, Immunoblot analysis of LILRB4 phosphorylation and SHP2 recruitment in the presence of SCG2. HEK293T cells were co-transfected with Flag-tagged wild-type or mutant LILRB4 plasmids (2 μg), SCG2 (300 ng), SHP2 (300 ng), and Lyn (100 ng). Cell lysates were IP using anti-Flag beads and analyzed by IB with indicated antibodies.
