Sema-3A signaling. (A) H28 cells were transfected as described in Figure 5. Seventy-two hours from transfection, cells were incubated with T cells that had been prelabeled with the fluorescent dye hydroethidine. Heterotypic cell clustering was measured by flow cytometry. The involvement of NP-1 in heterotypic cell clustering was studied by preincubating labeled allogenic resting T cells with blocking NP-1 antibodies (T cells + anti-NP-1) or with nonblocking NP-1 antibody (T cells + Control Ab). ^*P < .05, ANOVA, n = 3. Error bars indicate SEM. (B) T cells were stimulated for the indicated time points with anti-CD3 + anti-CD28 in the presence of Sema3A/Fc or Sema6A/Fc (100 ng/mL). Cell lysates were then immunoblotted with antibodies against phosphorylated ERK1/2 (anti-p-ERK1/2), p38 MAPK (anti-p-38), or phosphorylated MEK1 (anti-p-MEK1). The blots were stripped and reprobed for total ERK1, p38, or MEK1. (C) Jurkat cells were stimulated with anti-CD3 alone or with anti-CD28 in the presence of Sema3A/Fc (right) or Sema6A/Fc (left) (100 ng/mL) for 10 minutes and lysed. GTP-bound Rap1 and H-ras were detected with pulldown assays by using immobilized GST fusion proteins of RalGDS-RBD and Raf-RBD as described in “Materials and methods.” Western blot of total cell lysates with anti-Ras and anti-Rap1 antibody is also shown. Data are representative of 3 experiments.