Figure 6

Poly(ADP-ribosyl)ation by PARP-1 of HMGB1 lacking the C-terminal tail and of HMGB1 A-Box, but not HMGB1 B-Box, potentiates the inhibitory effects of HMGB1 on efferocytosis. (A) PARylation of full-length HMGB1 and HMGB1 domains by PARP-1. Full-length HMGB1-His, AC-HMGB1-His, A Box-His or B Box-His (500 μg/mL) were incubated with recombinant PARP-1 in a poly(ADP-ribosyl)ation reaction containing NAD and activated DNA for 30 min. The reactions were terminated with sample buffer and subjected to immunoblot analysis with antibodies to PAR or His. Representative gels are shown. Two additional independent experiments provided similar results. (B) Phagocytosis assays were performed after preincubation of apoptotic thymocytes with 1 μg/mL unmodified or PARylated HMGB1-His, AC-HMGB1-His, A Box-His or B Box-His for 2 h. The thymocytes were then washed before being added to macrophages. *P< 0.05 compared with control. ^#P< 0.05 compared with thymocytes treated with non-PARylated HMGB1 or HMGB1 domains.