Figure 3. RIP2 kinase inhibitors SB 203580 and PP2 suppress MDP–NOD2-induced activation of NF-κB.

All experiments were performed in duplicate, and similar results were obtained in at least two independent experiments. (A) HEK-293 cells were co-transfected with 0.1 μg of DNA encoding 3×κB luciferase reporter construct and 0.1 μg of pTK-RL plasmid DNA (encoding Renilla luciferase) plus 0.8 μg of empty pCMV5 vector (to bring the final amount of vector to 1.0 μg as used for all experiments) and the indicated amounts of plasmid DNA encoding HA-tagged NOD2. After 24 h, cells were incubated without (white bars) or with (black bars) 1 μM MDP. After 24 h the cells were lysed and NF-κB-dependent luciferase reporter gene expression was analysed. The error bars show the S.D. between duplicate experiments. (B) Effect of PP2 on the activity of RIP2 in vitro. RIP2 was assayed as described in the Experimental section at the indicated concentrations of PP2. The IC₅₀ value for inhibition by PP2 was 10 nM. (C) Experiments were performed as described for (A), using 1 ng of DNA encoding HA-NOD2 for transfection. At 1 h before stimulation with (black bars) or without (white bars) MDP the cells were incubated without (control) or with 10 μM SB 203580, 0.1 μM BIRB 0796 or 1 μM PP2. The error bars show the S.D. between duplicate experiments. (D) HEK-293 IL-1R cells were transfected with the 3× κB luciferase reporter construct and pTK-RL plasmid DNA as described in (A), treated with the indicated inhibitors as in (C) and stimulated with 5 ng/ml IL-1β instead of MDP. The error bars show the S.D. between duplicate experiments. (E) HEK-293 IL-1R cells were incubated without (control) or with 10 μM SB 203580, 0.1 μM BIRB 0796 or 1 μM PP2 1 h before stimulation with 5 ng/ml IL-1β for 20 min. Cells were lysed and 20 μg of extract protein was analysed by immunoblotting with antibodies recognizing phosphorylated TAB1 (phosphothreonine-431) and p38α MAPK as well as IκBα and GAPDH as a loading control. (F) RAW 264.7 cells were transiently co-transfected via electroporation with 50 μg of DNA encoding 3× κB luciferase reporter construct and 5 μg of pTK-RL plasmid DNA (encoding Renilla luciferase) (see the legend to Figure 1A). Cells were left to recover for 4 h in RPMI 1640 supplemented with 10% heat-inactivated FCS, and the medium was then replaced with RPMI 1640 without FCS. The cells were incubated for 60 min with (+) or without (−) 5 μM SB 203580 or 0.1 μM BIRB 0796, then stimulated with (+) or without (−) 1 μM MDP or 100 ng/ml LPS or left untreated. After 24 h, the cells were extracted in 0.5 ml of Passive Lysis Buffer and 20 μl aliquots of the extract were analysed for NF-κB-dependent luciferase reporter-gene expression. The results are given as means±S.D. for three different experiments, each performed in duplicate.