Ig domains 1 and 4 in LILRB2 are critical for Angptl2 binding and signal activation. (A) Representative flow cytometry plots showing Angptl2 binding to full-length, individual Ig domain, Ig1+2, or Ig3+4 of LILRB2 that were expressed on 293T cells; n = 3. (B) Summary of data from panel A. (C) Summary of Angptl2 binding abilities of wild-type (WT) and mutant LILRB2. Indicated mutations are described in supplemental Figure 3B. (C) Schematic of the H*G*Y*C motifs in Ig1 and Ig4 of LILRB2. (D) Summary of Angptl2 binding abilities of WT and mutant Ig1+2 LILRB2. (E) Representative flow cytometry plots showing Angptl2 binding to Ig1+2 and mutant LILRB2. (F) Representative flow cytometry plots showing Angptl2 binding to WT and mutant LILRB2. (G) Comparison of Angptl2, Angptl5, and HLA-G binding abilities of WT and mutant LILRB2. MHC-S indicates HLA-G binding sites; MHC-S1, R59A/Y61A; MHC-S2, W90A/D200A/N202A/Y205A; MHC-S1+2, R59A/Y61A/W90A/D200A/N202A/Y205A. (H) Summary of Angptl2-induced activation of the chimeric receptor reporter system by individual Ig domains, Ig1+2, or Ig3+4 of LILRB2. Indicated reporter cells were treated with 5 µg/mL coated GST-Angptl2 or polyclonal or monoclonal anti-LILRB2 antibodies. At least 3 independent experiments gave the similar results. (I) Summary of Angptl2-induced activation of the chimeric receptor reporter system by WT or mutant LILRB2. Reporter cells were treated with 10 µg/mL coated GST-Angptl2 or polyclonal or monoclonal anti-LILRB2 antibodies. At least 3 independent experiments were performed that gave the similar results. NC, negative control cells that do not express the chimeric receptors.