Figure 6.
Figure 6. Gal-9 binds to 4-1BB. (a) Binding of m4-1BB.Fc or mBTLA.Fc or control hIgG to plate-bound recombinant mGal-1, mGal-9, or m4-1BBL, as measured by optical density. (b) Flow cytometry detection of binding of recombinant m4-1BBL, mGal-9, or mGal-3 to saturating amounts of m4-1BB.Fc coated on protein G beads. Shaded histograms, binding to control hIgG (Fc fragment)–coated beads. (c) SDS-PAGE of coimmunoprecipitation of h/m4-1BBL, h/mGal-9, or h/mGal-4 with h4-1BB.Fc (left) or m4-1BB.Fc (right). The observed molecular masses (kilodaltons) were h/m4-1BB.Fc, 55–58; h4-1BBL, 20; m4-1BBL, 41–44; m/hGal-9, 36; and m/hGal-4, 36. Data in a–c are representative of at least three different experiments each. (d and e) Binding response of increasing concentrations (0.004–0.312 µM) of hGal-9 (d) or mGal-9 (e) to immobilized h4-1BB.Fc (d) or m4-1BB.Fc (e), as measured by SPR. Values represent the mean of three independent measurements. The response shown is reference-subtracted (unrelated Fc protein). (f) Flow analysis of competition between m4-1BBL/anti–4-1BB antibodies and mGal-9 for binding to m4-1BB.Fc. m4-1BB.Fc–coated protein G beads were incubated either with m4-1BBL (top left) or mGal-9 (bottom left) alone. For competition between m4-1BBL and mGal-9, m4-1BB.Fc–coated beads were first incubated with m4-1BBL (second left) or mGal-9 (middle). The beads were washed and incubated further with mGal-9 (second left) or m4-1BBL (middle). Binding of m4-1BBL (top) or mGal-9 (bottom) was detected using different fluorescent-labeled antibodies and analyzed by flow. Shaded histogram, binding of respective detection antibody isotype. For competition between anti–4-1BB antibodies and mGal-9, m4-1BB.Fc–coated beads were first incubated with agonistic 4-1BB antibodies, clone 3H3 (second right) or 1D8 (right). The beads were washed and further incubated with mGal-9. Open black histograms, mGal-9 binding without anti-4-1BB antibodies. Gray-shaded histograms, mGal-9 binding after anti-4-1BB antibodies. Black-shaded, beads with no mGal-9. Data are representative of two different experiments.

Gal-9 binds to 4-1BB. (a) Binding of m4-1BB.Fc or mBTLA.Fc or control hIgG to plate-bound recombinant mGal-1, mGal-9, or m4-1BBL, as measured by optical density. (b) Flow cytometry detection of binding of recombinant m4-1BBL, mGal-9, or mGal-3 to saturating amounts of m4-1BB.Fc coated on protein G beads. Shaded histograms, binding to control hIgG (Fc fragment)–coated beads. (c) SDS-PAGE of coimmunoprecipitation of h/m4-1BBL, h/mGal-9, or h/mGal-4 with h4-1BB.Fc (left) or m4-1BB.Fc (right). The observed molecular masses (kilodaltons) were h/m4-1BB.Fc, 55–58; h4-1BBL, 20; m4-1BBL, 41–44; m/hGal-9, 36; and m/hGal-4, 36. Data in a–c are representative of at least three different experiments each. (d and e) Binding response of increasing concentrations (0.004–0.312 µM) of hGal-9 (d) or mGal-9 (e) to immobilized h4-1BB.Fc (d) or m4-1BB.Fc (e), as measured by SPR. Values represent the mean of three independent measurements. The response shown is reference-subtracted (unrelated Fc protein). (f) Flow analysis of competition between m4-1BBL/anti–4-1BB antibodies and mGal-9 for binding to m4-1BB.Fc. m4-1BB.Fc–coated protein G beads were incubated either with m4-1BBL (top left) or mGal-9 (bottom left) alone. For competition between m4-1BBL and mGal-9, m4-1BB.Fc–coated beads were first incubated with m4-1BBL (second left) or mGal-9 (middle). The beads were washed and incubated further with mGal-9 (second left) or m4-1BBL (middle). Binding of m4-1BBL (top) or mGal-9 (bottom) was detected using different fluorescent-labeled antibodies and analyzed by flow. Shaded histogram, binding of respective detection antibody isotype. For competition between anti–4-1BB antibodies and mGal-9, m4-1BB.Fc–coated beads were first incubated with agonistic 4-1BB antibodies, clone 3H3 (second right) or 1D8 (right). The beads were washed and further incubated with mGal-9. Open black histograms, mGal-9 binding without anti-4-1BB antibodies. Gray-shaded histograms, mGal-9 binding after anti-4-1BB antibodies. Black-shaded, beads with no mGal-9. Data are representative of two different experiments.

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