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. 2017 Sep 20;8(47):82740–82753. doi: 10.18632/oncotarget.21098

Figure 7. B7x does not directly interact with Neuropilin-1.

Figure 7

(A) Human NRP1-hIgG1 (Sino Biological) was immobilized on sensors. Recombinant human vEGF (Peprotech) from concentrations of 0.025 mg/mL, 0.05 mg/mL and 0.1 mg/mL were challenged. The responses of vEGF were concentration dependent (dashed lines). Recombinant human B7x (R&D) was not responsive even at the concentrations of 0.25 mg/mL and 0.5 mg/mL (solid curve). (B) Human B7x-hIgG1 (R&D) was immobilized and recombinant human NRP1 (R&D) at the concentrations of 0.25 mg/mL and 0.5 mg/mL were challenged. (C) Human B7x-hIgG1 (Sino Bilogical) was immobilized and recombinant human NRP1 (R&D) at the concentrations of 0.25 mg/mL and 0.5 mg/mL were challenged. (D) Human Sema3a-hIgG1 (R&D) was immobilized on sensors. Rh-NRP1 (R&D) from concentrations of 0.025 mg/mL, 0.05 mg/mL and 0.1 mg/mL were challenged. The responses of vEGF were concentration dependent (dashed lines). Recombinant human B7x (R&D) was not responsive even at the concentrations of 0.25 mg/mL and 0.5 mg/mL (solid curve). The blue and red arrows indicate the starts of the association and dissociation, respectively. (E) 3T3 cells expressing Neuropilin-1 were incubated with either 10 ug/mL hIg control (shaded), 1 ug/mL (solid black line) or 10 ug/mL Sema3a-hIgG1, B7x-Ig, or B7x-Ig (IgV1 domain) protein (dashed black line) and then stained with APC-conjugated anti-human IgG Fc antibody to assess binding activity by FACS. (F) Incubation of labeled PD-1 with its ligands PD-L1 and PD-L2 resulted in 18% and 24% co-localization. Confirmed non-binding partners, PD-1 and B7x, resulted in approximately 4% non-specific binding, while NRP1 and B7x incubation led to approximately 3% co-localization. Data are representative of two separate experiments.