Sema-3A inhibited CD3 plus CD28-mediated IL-2 transcription and expression and transactivation of known IL-2 transcription factors. (A) Jurkat T cells were transiently transfected with the reporter construct pIL-2-luc (10 μg). Forty hours later, 5 × 105 cells per sample were cultured for 8 hours with either anti-CD3 + anti-CD28 alone or with increasing concentrations of recombinant human Sema-3A (Sema3A/Fc), and luciferase activity was measured. *P < .05 versus none, ANOVA, n = 4. Error bars indicate SEM. (B) Jurkat cells were cultured for 3 hours with anti-CD3 + anti-CD28 in the presence of Sema3A/Fc or Sema6A/Fc (100 ng/mL), whole-cell extracts were prepared, and expression of transcription factors was determined by immunoblotting with antibodies specific for NFATp, c-Fos, and actin. The whole-cell extracts was also immunoblotted using anti-phospho-c-Jun and anti-c-Jun antibodies. (C) Whole-cell extracts from Jurkat cells, treated as described in panel B for the indicated times, were prepared and immunoblotted with an anti-IκBα antibody and reprobed with an anti-JNK1 antibody to control for loading. Data are representative of 2 experiments.
