Figure 4.

CD28-mediated activation of the c-fos promoter requires prolines 187 and 190 and Lck. (A) CD28 requires prolines 187 and 190 to activate the c-fos promoter. Wild-type and mutated mCD28 (P187,190A) was transiently cotransfected into Jurkat cells with a c-fos reporter plasmid. Anti-CD28 mAb (37.51) was added to the media 10–12 h after electroporation, and cells were harvested and assayed for luciferase activity 24 h later. CD28 surface expression was verified by flow cytometry, and transfections were normalized using a renilla luciferase reporter plasmid. Data is shown as fold activation compared with unstimulated cells transfected with the c-fos reporter plasmid alone. (B) CD28 stimulation of the c-fos reporter requires Lck. Wild-type mCD28 was cotransfected with the c-fos reporter plasmid into JCaM1.6 cells, which lack Lck expression. Experiments were performed as described in A. In this experiment, PMA stimulation of the fos-luciferase (fos-luc) reporter was used to control for transfection efficiency. All data are representative of at least three independent trials.