Immunostimulatory effects of THP are dependent on p38-, ERK-1/2–MAPK kinase and NF-κB signaling. Immature human DCs (A) or murine macrophages (B) were incubated with THP, LPS, or medium. Subsequently, phospho-p38 (p-p38) and phospho–ERK-1/2 (p-ERK) were determined by immunoblotting; p38 and ERK-2, respectively, were detected from stripped membranes. Blots are representative of 4 independent experiments. (C) Immature DCs were incubated with THP, LPS, or medium, and phospho-Akt (p-Akt) as well as total Akt were determined from whole cell lysates by immunoblotting. Data are representative of 3 independent experiments. (D) Immature human DCs preincubated with or without the indicated MAPK inhibitors were exposed to THP, LPS, or medium. Cell-free supernatants were collected after 18 hours and were analyzed for TNF-α by ELISA. Data are expressed as mean ± SEM of 4 different donor combinations. *Significantly different from the values for stimulated control; P < 0.05. (E) Immature DCs were incubated with THP, LPS, or medium, and immunoblotting was performed from whole-cell lysates using Abs against IκB-α and ERK-2. (F) THP, LPS, or medium was added to immature human DCs. Oligonucleotides labeled with ³²P, containing a NF-κB consensus sequence, were incubated with nuclear extracts followed by nondenaturing gel electrophoresis. Similar results were obtained in 2 independent experiments. (G) Immature human DCs preincubated with or without the NF-κB inhibitor SN50 were exposed to THP, LPS, or medium. Cell-free supernatants were collected after 18 hours and were analyzed for cytokines by ELISA. Data are representative of 4 independent experiments. *Significantly different from the values for stimulated control; P < 0.05.