Expression of KDR on mitogen-activated CD4⁺ and CD8⁺ T cells. CD4⁺ and CD8⁺ T cells were purified from human blood by positive selection, and were stimulated with anti-CD3/anti-CD28 (as outlined in “Leukocyte isolation”) for 72 hours or as indicated. The expression of KDR was evaluated by FACS (A), by real-time PCR (B), by Western blot analysis (C), and by confocal microscopy (D). In panel A, the difference (delta, Δ) in mean fluorescence staining (experimental minus isotype control) is shown within each FACS plot. In panel B, the relative fold induction in KDR mRNA expression in 6- and 12-hour mitogen-activated CD4⁺ and CD8⁺ T cells versus untreated cells (NT) is shown. Panel C illustrates a representative Western blot of KDR in unactivated (lane 1) and 12-hour mitogen-activated T cells (lane 2). Panel D shows representative confocal microscopy images of 72-hour anti-CD3/anti-CD28-activated CD4⁺ T cells stained for CD3 and KDR. Approximaetly 20% of all activated T cells in our cultures express KDR (as illustrated) and the remainder of the T cells in the cultures have nondetectable or low levels of expression (not shown). All experiments are representative of n ≥ 5 per condition (A-B) and n = 3 (C).