Plasmids used for the M2H assays were based on the CheckMate mammalian two-hybrid system (Promega). The TIR domain of mouse MyD88 (amino acids 158 to 296) was cloned into the Gal4 vector (pBIND) and VP16 vector (pACT), full-length mouse MyD88 was cloned into pACT, the TIR domain of mouse TLR9 (amino acids 867 to 1032) was cloned into pBind, and mouse TIRAP (amino acids 1 to 241) was cloned into pBind. HEK293T cells were transiently transfected with bait and prey plasmids in 96-well format using Lipofectamine 2000, along with a Gal4-driven firefly luciferase reporter plasmid (pGL5-luc, Promega) and Renilla luciferase control vector (Promega). DMSO or compound was added to cells 7 hours after transfection. Cells were harvested 13 hours later, and luciferase activity was determined using the dual-luciferase kit (Promega). Firefly luciferase activity values were normalized to Renilla luciferase activity.

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