Fig. 2.

HMGB1 promotes binding of soluble CD52-Fc to Siglec-10. Binding of CD52-Fc to recombinant HMGB1 and Siglec-10-Fc is shown. Proteins immobilized in flat bottom 96-well microtiter plate wells were as follows: CD52-Fc (at 10 μg/mL) (A and D), Siglec-10-Fc (at 10 μg/mL) (B), HMGB1, HMGB1 Box B, and HMGB1 Box A (each at 20 μg/mL) (C). After blocking the plate with binding buffer, different proteins as indicated were added (50 μL per well, 10 μg/mL) to triplicate wells, and binding was detected as described in SI Appendix, SI Materials and Methods. Results are mean ± SD of three separate experiments. (E) CD52-Fc or Fc (each 20 μg/mL) and Siglec-10-Fc (10 μg/mL) were incubated separately or together with HMGB1 or HSPs 70 or 90 (each 20 μg/mL) overnight at 4 °C. CD52-Fc (or Fc) was then precipitated (P) with Strep-Tactin beads and the pellet fractionated by SDS/PAGE and immunoblotted (IB) with anti-Siglec-10 antibody.