FIG. 1.

Preferential activation of the Ras-MAP kinase signaling cascade in CD45RO⁺ T cells (part 1). (A) CD45ABC⁺ and CD45RO⁺ TCR⁺ CD3⁺ CD4⁺ BW5147 T-cell clones, in the order indicated in panel C, were transfected with an Elk-1–GAL4 transactivator and a 5×GAL4-luciferase reporter at a ratio of 1:10 and stimulated for 24 h with plate-bound anti-D10 TCR MAb 5A (10 μg/ml). The relative activity of the reporter is expressed as fold induction over reporter transactivation in unstimulated cells. (B) CD45ABC⁺ and CD45RO⁺ cells were transfected with the Elk-1–GAL4–5×GAL4–luciferase reporter, cotransfected with 50 ng of RasN17 or a matched empty vector, and stimulated with MAb 5A. Luciferase activities were normalized against an internal control (pRL-CMV). (C) Western blot analysis of whole-cell lysates of CD45ABC⁺ (clones 17.11, 17.4, and 23.3) and CD45RO⁺ (clones 19.9 and 18.16) cells stained with anti-CD45 MAb 30-F11. (D) CD3⁺ CD4⁺ CD45RO⁺ BW5147 cells were transfected with Elk-1–GAL4 (or the GAL4 DNA binding domain [GAL4dbd] alone) and a 5×GAL4-luciferase reporter and cotransfected with either the empty vector (Emp. vec.) or an expression plasmid(s) containing the dominant negative mutant forms of Erk or Raf (RafN4) under the control of a constitutive promoter. At 24 h posttransfection, cells were left unstimulated or were stimulated for another 24 h with an anti-TCR MAb. Luciferase activity (in relative luciferase units [RLU]) was normalized against the protein concentration of the cell lysates and expressed as a percentage of the control stimulation. DMSO, dimethyl sulfoxide. (E) Transactivation of the Elk-1–GAL4–5×GAL4–luciferase reporter system in CD45-positive and CD45-negative BW cells upon stimulation with an anti-TCR MAb (top) or 20% fetal calf serum (FCS) following overnight starvation (bottom). The data shown are representative of at least two independent experiments.