Figure 4. Effect of hydrogen peroxide and Ro 318220 on the phosphorylation of ERK8.

(A) HEK-293 cells were transfected with vectors expressing either wild-type (WT) HA–ERK8 or HA–ERK8[D154A] as described in the Materials and methods section. At 36 h post-transfection, the cells were exposed for 10 or 30 min to 1 mM hydrogen peroxide, and lysed. The lysates (60 μg) were denatured in SDS, subjected to SDS/PAGE and, after transfer to PVDF membranes, immunoblotted with the phosphospecific antibody that recognizes the phosphorylated Thr-Glu-Tyr motifs of ERK1, ERK2 and ERK8, and an antibody that recognizes phosphorylated and unphosphorylated ERK8 equally well. (B) HA–ERK8 in 30 mg of cell lysate protein was immunoprecipitated from HEK-293 cells exposed to hydrogen peroxide (lower panel) or left unstimulated (upper panel) was digested with trypsin and analysed by LC–MS. The extracted ion chromatograms for the doubly charged peptide ions ([M−2H]²⁻) representing the singly phosphorylated (m/z=1162–1163) and doubly phosphorylated (m/z=1202–1203) forms of the peptide comprising Ser-161–Arg-182 are shown. (C) HEK-293 cells transfected with HA–ERK8 as in (A) were incubated for 1 h with the indicated concentrations of Ro 318220, then exposed for 10 or 30 min to 1 mM hydrogen peroxide and lysed. Cell lysate (45 μg) was then analysed as in (A). (D) Wild-type GST–ERK8 and GST–ERK2 (both from E. coli) were assayed in vitro using MBP as substrate at the indicated concentrations of Ro 318220.