Rap1 mediates the immunoinhibitory effects of Sema-3A. (A-B) Jurkat T cells were transfected with an empty vector (pcDNA3.1) or a vector containing cDNA of a dominant-negative Rap1 (Rap1N17). Then, stable transfectants were stimulated with anti-CD3 + anti-CD28 in the absence or presence of Sema3A/Fc (100 ng/mL) for 10 minutes and lysed. Cell lysates were electrophoresed and blotted with anti-p-ERK1/2 antibody. The blots were stripped and reprobed for total ERK1 (A). After 24 hours, the supernatants were harvested for IL-2 measurement by ELISA. ^*P ≤ .05, ANOVA, n = 3. Error bars indicate SEM. (C) Control and anti-CD3 and anti-CD28-stimulated T cells (10⁷ cells per test) were either untreated or treated with Sema3A/Fc (100 ng/mL) for 5 minutes, and the lysates were then prepared. Equivalent amounts of whole-cell lysates were either immunoprecipitated with anti-Ras antibody and immunoblotted with anti-Raf-1 antibody (right) or immunoprecipitated with anti-Rap-1 antibody and immunoblotted with anti-Raf-1 antibody (left). Western blot of immunoprecipitated cell lysates with anti-Rap1 and anti-Ras antibody is also shown. Data are representative of 3 experiments.