Figure 2. RIP2 inhibitor SB 203580 decreases the protein expression levels of WT-RIP2, but not KI-RIP2, in transfected HEK-293 cells.

All experiments were carried out at least twice with similar results. (A) Effect of SB 203580 and BIRB 0796 on the activity of RIP2 in vitro. RIP2 was assayed as described in the Experimental section at the indicated concentrations of SB 203580 or BIRB 0796. The IC₅₀ value for inhibition by SB 203580 was 43 nM. (B) Cells were transfected with DNA constructs encoding HA-tagged versions of either WT-RIP2 or KI-RIP2 (1 μg of plasmid RIP2 DNA). After 24 h the cells were incubated for a further 24 h with (+) or without (−) 10 μM SB 203580, then extracted with standard lysis buffer. The level of transfected RIP2 in the extract was analysed by immunoblotting with an anti-HA antibody. (C) Cells were transfected for 48 h with a construct encoding HA-tagged WT-RIP2 or RIP2[T95M] (1 μg of plasmid RIP2 DNA). The cells were incubated with 10 μM SB 203580 or 0.1 μM BIRB 0796 for 1 h before the end of transfection period, lysed, and 20 μg of extract protein was analysed by immunoblotting with an anti-HA antibody. The extracts were also immunoblotted with antibodies that recognize GAPDH as a loading control.