Inhibition of SEB Intoxication by a MyD88 Mimetic Figure 2. Structure of monomeric (Compound1) and dimeric (EM-163) mimetics of the BB-loop in the TIR domain of MyD88. doi:10.1371/journal.pone.0040773.g002 Increase in Accumulation of MyD88 in the Presence of EM-163 Our earlier results demonstrated that SEB stimulation induces de novo synthesis of MyD88 in primary cells compared to non- stimulated cells [12,19]. We examined the effects of EM-163 on levels of MyD88 protein in SEB-stimulated and unstimulated primary monocytes. Purified monocytes were treated with SEB in the presence or absence of EM-163 and levels of MyD88 were analyzed by confocal microscopy. Consistent with the previously reported up-regulation of MyD88, monocytes stimulated with SEB caused an increase in intracellular accumulation of MyD88 (Figure 4). An increase in MyD88 levels was likewise seen with SEB stimulation in the presence of EM-163 both at 1 hr and 4 hr. However, an increase in MyD88 levels was observed in some Figure 3. EM-163 inhibits TIR-TIR interaction. A microarray-based binding assay was optimized to measure compound EM-163 binding to MyD88. Nitrocellulose coated slides were spotted in quadruplicate with anti-MyD88 antibody and MyD88 TIR domain monomer proteins using the ArrayJet Marathon printer. Biotinlyated MyD88 (25 mg/ml) was pre-incubated with compound (100 mg/ml and 50 mg/ml), dimethylsulfoxide as control, or left without pre-incubation. Streptavidin- labeled with alexa fluor 647, was added for 1 h at room temperture to detect MyD88 binding interaction. Slides analyzed using the Genepix 4000B. Data represent four similar experiments. doi:10.1371/journal.pone.0040773.g003 PLoS ONE | www.plosone.org 5 July 2012 | Volume 7 | Issue 7 | e40773