Figure 2.

α-Dma detects Vav1 upon CD28 costimulation. (A) CD28WT, CD28Del30, and CD28Neg cells (1.5 × 10⁷) were stimulated with 0.5 × 10⁷ 5–3.1-B7 (+), or 5–3.1 (−) cells for the indicated times. Lysates were immunoprecipitated with α-Dma and immunoblotted with α-Vav1. Immunoblot for comparable protein content in lysates is shown (bottom). This is one representative of five independent experiments. (B) CD28WT cells were stimulated as in A for the indicated times. Lysates were immunoprecipitated with α-Dma in parallel with α−Vav1, α-Lck, α-Grb-2, and α-Akt antibodies. Immunoblots were with the indicated antibodies. (C) Vav1-deficient Jurkat cells (JVav) and Jurkat cells were stimulated with 5–3.1 or 5–3.1-B7 cells for the indicated times and lysates were treated as in A. Controls for Vav1 were done by α-Vav1 immunoblot (bottom). (D) CD28WT and CD28Del30 cell lines (1.5 × 10⁷) were stimulated with 0.5 μg/ml SEB prepulsed 5–3.1-B7 cells (0.5 × 10⁷) for the indicated times. Lysates were treated as in A and immunoblotted with α-Vav1. (bottom) Comparable Vav1 content in lysates. One experiment is shown of two giving similar results. (E) 2 × 10⁷ cultured T cells were pretreated for 1 h with 0.3 mM MTA before stimulation with 0.5 × 10⁷ 5–3.1-B7 cells. Cell lysates were reacted to α-Dma as in A, followed by α-Vav1 immunoblotting. Similar results were obtained in two experiments. (F) CD28WT cells treated with or without PP2 (10 μM) were stimulated for 3 or 45 min and processed as in A. 10 μM of PP2 strongly inhibited B7-induced Vav1 tyrosine phosphorylation (not depicted).