Figure 3
PARylated HMGB1 binds to PS and RAGE with higher affinity than non-PARylated HMGB1. (A) After being precoated with the integrins αVβ3 or αVβ5, 96-well plates were incubated with 1 μg/mL HMGB1 or PARylated HMGB1 for 2 h. The plates were then washed and bound HMGB1 was quantified by ELISA, as described in Materials and Methods. **P < 0.01 compared with HMGB1. (B) Gas6 and MFG-E8-precoated 96-well plates were incubated with 1 μg/mL HMGB1 or PARylated HMGB1 for 2 h. The plates were then washed and bound HMGB1 was quantified by ELISA. *P < 0.05 and **P < 0.01 compared with HMGB1. (C) Recombinant RAGE dissolved in PBS or PS dissolved in methanol was incubated in 96-well plates. The plates were then incubated with 1 μg/mL HMGB1 or PARylated HMGB1 for 2 h. Bound HMGB1 was quantified by ELISA. *P < 0.05 compared with HMGB1. Assays were done in triplicate.