Fig 1: Binding characteristics of RbPLX7. A, SPR-binding sensogram showing binding kinetics of the RbPLX7 antibody (1 µg/ml) to the extracellular domain (20–535) of human (black line), cynomolgus (green line), and mouse Plexin-B1 (gray line) (10 µg/ml). B, kinetic-binding curves obtained by SPR multicycle kinetics titration of recombinant human Plexin-B1 (20–535) on the sensor-immobilized RbPLX7 antibody. Individual sensograms (from top to bottom) correspond to 100, 50, 30, 20, 10, 5, and 2.5 nM of Plexin-B1 (20–535). C, binding of RbPLX7 to full-length human Plexin-B1, -B2, and -B3 expressed in Expi293 cells as analyzed by flow cytometry. Human IgG1 represents the isotype control used in each assay. D, biolayer interferometry sensogram showing the binding of Sema4D (50 nM) to human recombinant Plexin-B1 (20–535) (85 nM) in the presence (black sensogram) and absence (gray sensogram) of RbPLX7 (50 nM). Injection points of proteins or buffer are indicated. Representative examples of at least three (Fig. 1, A, B, and D) or 2 (Fig. 1C) independent experiments (biological replicates) are shown. MFI, median fluorescence intensity; Sema4D, semaphorin 4D; SPR, surface plasmon resonance.
Fig 2: RbPLX7 blocks Sema4D-induced inhibition of osteoblast differentiation and mineralization.A, human osteoblasts were grown in osteoblast growth medium (control) or in osteogenic medium (OST) to induce differentiation and mineralization. Osteoblasts were then exposed to 150 nM of Sema4D in osteogenic medium without or in combination with 150 nM anti–Plexin-B1 antibody RbPLX7 (RbPLX7) or the corresponding IgG isotype control antibody (IgG). Osteoblast differentiation was determined after 7 days via measurement of alkaline phosphatase (ALP) activity, and osteoblast mineralization was analyzed after 21 days via Alizarin Red S staining. The scale bar represents 100 µm. B and C, quantification of the results in (A). Shown is a representative example of 3 independent experiments (biological replicates). Graphs depict mean values ± s.d. Sema4D, semaphorin 4D.
Fig 3: RbPLX7 blocks Sema4D-induced COS-7 cell collapse.A, COS-7 cells were transfected with cDNA encoding human Plexin-B1-FLAG (human Plexin-B1), mouse Plexin-B1-FLAG (mouse Plexin-B1), or human Plexin-B2-FLAG (human Plexin-B2). Forty eight hours after cDNA transfection, cells were treated with RbPLX7 or IgG isotype control (10 nM for Plexin-B1–expressing cells and 150 nM for Plexin-B2–expressing cells), followed by treatment without or with Sema4D (50 nM for Plexin-B1–expressing cells and 150 nM for Plexin-B2–expressing cells). Shown are representative fluorescence images of immunostainings using anti-FLAG antibodies (green). White arrows indicate examples of collapsed cells. The scale bar represents 50 µm. B and C, COS-7 cells were transfected as described in (A), treated with (B) RbPLX7, or (C) IgG isotype control at the indicated concentrations followed by Sema4D, and cellular collapse was determined as described in Experimental procedures. Shown are representative examples of two independent experiments (biological replicates). Graphs depict mean values ± s.d. Sema4D, semaphorin 4D.
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