Figure 4.

MDL 28, 842 inhibits Vav1 R-methylation and IL-2 production. (A) 1.5 × 10⁷ CD28WT cells were pretreated for 1 h with 0.1 and 1 μM MDL and stimulated for 40 min with 0.5 × 10⁷ SEB-pulsed 5–3.1-B7 cells, as in Fig. 2 B. Lysates were processed, immunoprecipitated with α-Dma, and Vav1 was detected as in Fig. 2 B. Numbers indicate relative amounts of methylated Vav1 signal after normalization for protein (bottom). Similar results were obtained in two experiments. (B) 5 × 10⁵ CD28 WT cells (triplicates) were treated for 1 h with 0.1, 1, and 5 μM of MDL. After washing, they were stimulated for 3.5 h with 10⁵ 5–3.1-B7 cells pulsed with SEB (0.5 μg/ml). Brefeldin A (10 μg/ml) was added after 1 h of incubation. Cells were treated as described in Materials and methods for IL-2 FACS analysis. Similar results were obtained in four experiments.