SUPPLEMENTARY FIGURE 1. A, rTMD123 can interact with HMGB1 in the aorta. Fifty-μg rTMD123 protein (tagged with c-myc), rabbit anti-HMGB1 antibody, and protein A beads were added in the aortic homogenates (obtained on day 3) overnight at 4C. Rabbit IgG antibody (IgG) was served as a negative control. Beads were washed thrice with PBS containing 0.05% Tween-20 and the immune precipitates were analyzed by western blot. The result is typical of those obtained in 3 independent experiments. B, rTMD123 inhibits HMGB1 binding to THP-1 cells in a concentration-dependent manner. THP-1 cells were co-incubated with recombinant HMGB-1 protein and rTMD123 for 30 min at 4C. Cells were washed, stained with rabbit-anti HMGB-1 antibody for 30 min at 4C, and were stained with Alexa Fluor 488-conjugated goat anti-rabbit antibody for 30 min at 4C. Cells were analyzed by FACS. The geometric mean fluorescence intensity was determined by WinMDI 2.9 software (n=3). (**P<0.01 compared with untreated group. #P<0.05 compared with HMGB1-treated group.)

IgG HMGB1

IB: c-myc

IP

BA

HMGB1 (nM) 0 32 32 32

0 0 8 16rTMD123 (nM)

Mea

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cenc

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tens

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25

30

35

40