FIG. 2.

Preferential activation of the Ras-Raf-Mek signaling cascade in CD45RO⁺ T cells (part 2). Transactivation of the reporter construct pB4X-CAT in CD45-negative (CD45NEG), CD45ABC⁺ (clones 17.11, 17.4, and 23.3), and CD45RO⁺ (clones 19.9 and 18.16) TCR⁺ CD3⁺ CD4⁺ BW5147 T cells. Cells in the indicated order were transfected with the CAT reporter and stimulated for 48 h with the antigen (Ag; conalbumin peptide CA37 [100 μg/ml] or PMA [50 ng/ml]). The relative activity of the reporter is expressed as the ratio of stimulation with the peptide to stimulation with PMA. (B) Autoradiogram of thin-layer chromatography and conversion of acetylated [¹⁴C]chloramphenicol in a CAT assay of TCR⁺ CD3⁺ CD4⁺ CD45⁺ T cells transiently transfected with pB4X-CAT alone (control, dimethyl sulfoxide [DMSO], and PD98059) or transfected with pB4X-CAT and cotransfected with either an empty vector control (pCGN) or an expression plasmid containing dominant negative mutant Raf (RafN4). Posttransfection, cells were nonstimulated, stimulated for 48 h with CA37 without additional treatment, or stimulated and simultaneously treated with dimethyl sulfoxide or 10 μM Mek1 inhibitor PD98058. APCs, antigen-presenting cells.