Figure 2. Absence of the C-terminal acidic tail abolishes the inhibitory effect of HMGB1 on efferocytosis-induced Erk phosphorylation and Rac-1 activation.

(A) Peritoneal macrophages were washed with serum-free medium and incubated in serum-free media for 1 h followed by exposure for 1 h to 1 μg/ml BSA (control), full-length HMGB1, or ΔC-HMGB1 in serum-free media. The macrophages were washed and incubated with RPMI-1640 medium with 1% FBS, with or without apoptotic neutrophils. After 15 min, the cells were washed, and cell extracts from the macrophages were prepared, Western blotting was performed to determine the levels of phosphorylated (p-Erk) and total Erk. A representative gel as well as mean ± sd of phosphorylated Erk/total Erk ratios for each condition using optical band intensity measurements from 3 independent experiments are shown. (B) Phagocytosis assays were performed for 15 min as described above. The macrophages were then washed 3 times and cell lysates prepared to determine Rac-1 activation. A representative gel as well as mean ± sd of activated/total Rac-1 ratios for each condition using optical band intensity measurements from 3 independent experiments are shown; *P < 0.05 versus control.