CDDO-Me inhibits ERK1/2 activation–phosphorylation.

(A) Western blot analysis of U937 cells revealed inhibition of ERK1/2 phosphorylation (pERK1/2) after 1 μM CDDO-Me treatment for 3 hours but no effect on ERK1/2 protein levels (ERK1/2). The intensity of the bands was quantitated by densitometry and expressed as a ratio of pERK2/ERK2 relative to the control value. (B) K562 cells were treated in vivo for 4 hours with 1 μM CDDO-Me, and lysate of these cells was used in the in vitro assay (lanes 1-3). As a negative control, a lysate containing inactive ERK1/2 was used in the assay (after 4-hour treatment of intact K562 cells with 10 μM MEK inhibitor PD98059). Phosphorylation of ERK substrate MBP was observed using an anti–phospho-MBP antibody after SDS-PAGE. In a parallel experiment, active ERK was immunoprecipitated from K562 cells treated with 0.1, 1, and 10 μM of CDDO-Me in vitro, and MBP was added as substrate with a cocktail of kinase inhibitors (lanes 4-7). (C) Active ERK was immunoprecipitated from HL-60 cells, and kinase activity was determined by MBP phosphorylation. The amount of ERK2 immunoprecipitated from each sample was determined by using anti-ERK2 antibody.