Figure 8. Expression of DUSP2 inhibits the ERK3 and ERK4-mediated phosphorylation of MK5.

NCI-H1299 cells were co-transfected with expression plasmids encoding either myc-tagged ERK4 (a and b) or myc-tagged ERK3 (C and D) together with an expression vector encoding an EGFP-MK5 fusion protein and either an empty expression vector or plasmids encoding either wild-type DUSP2 or a KIM mutant of DUSP2 as indicated. After 24 h, the cells were lysed and MK5 phosphorylated at Thr-182 was detected by Western-blotting using a phospho-specific anti-Thr182 MK5 antibody (a and c). The expression of MK5, ERK3, ERK4 and either wild-type DUSP2 or the DUSP2 KIM mutant were verified by Western blotting of the cell lysates using appropriate antibodies (bottom panels in a and c). Unprocessed original scans of the blots are shown in Supplementary Fig. 1. The data in a and c and two additional replicate experiments were then quantified using the Odyssey infrared imaging System. The relative intensity of the p-Thr182 MK5 bands are calculated using the band intensity from cells transfected with EGFP-MK5 alone as the reference and mean values are presented with associated errors (b and d).