T-cell activation via the RAS/MEK/ERK pathway is memory stage–dependent, and suppressed by MEK inhibition. (A) PBMCs were stimulated with PMA:ionomycin with ERK1/2 phosphorylation assessed within CD4⁺ and CD8⁺ T-cell maturation subsets. (B) Aggregate results (from n = 10 healthy donors) depicting ERK1/2 MFI within maturation subsets. MFI, mean fluorescence intensity. (C) Expression of RAS/MEK/ERK pathway members in CD8⁺ T cells by western blotting and collective results with densitometry values (from n = 3 healthy donors) standardized relative to expression in naive T cells. (D) ERK1/2 phosphorylation within PMA:ionomycin-activated CD4⁺ and CD8⁺ T cells with U0126 or selumetinib analyzed by phosphoflow cytometry. (E) Confirmation of dose-dependent inhibition of PMA:ionomycin-induced activation of purified CD3⁺ human T cells by U0126 by western blotting.