Dominant-negative Rap (Rap1A17) inhibits β-selection of Rag2^−/− DN cells in vitro. (A) Ba/F3 cell line was infected with empty MIG (□) or MIG containing SPA-1 (), Rap1N17 (), or Rap1A17 (■). The GFP⁺ cells were sorted, cultured in the presence of interleukin-3, and lysed. Rap1GTP was assessed by pull-down assay, and Rap1 activation indices are indicated (top). Aliquots of the cells were fluorescence-labeled and cultured in the absence or presence of interleukin-3 or PMA on fibronectin-coated plates for 30 minutes, then the fluorescence of cells adherent to fibronectin was examined (bottom). Means and SE of triplicate cultures are indicated. (B) Fetal thymocytes (FT) from Rag2^−/− mice (E17) were infected with empty MIG or MIG containing SPA-1 or Rap1A17 and cultured on TSt4/DLL1 stroma cells (top). One day later, 10 μg/mL anti-CD3ϵ antibody was added. The cells were harvested on days 1 and 6. Mean viable cell recoveries and SE of GFP⁺ cells at day 6 versus day 1 in 5 independent experiments are indicated (bottom). (C) The cells at day 6 in panel B were 2-color stained with anti-CD4 and anti-CD8; the representative profiles in a GFP⁺ gate are shown (top). The mean proportions and SE of DN (□), DP (■), CD4 SP (), and CD8 SP () cells in 5 independent experiments are indicated (bottom). *P < .01 compared with the proportions of corresponding subsets in vector control group.