Rap activation mediates the pre-TCR signal in a DN cell line. (A) SCID.adh cells were infected with empty MIN or MIN containing SPA-1 or Rap1N17, and NGFR⁺ cells were sorted. The cells were then infected with empty MIG or MIG containing CD8:CD3ϵ and cultured. Sixteen hours later the cells were harvested, lysed, and immunoblotted with the indicated antibodies (left). RapGTPs were detected by a pull-down assay. Similar results were obtained in 2 independent analyses. Twenty-eight hours after the culture, the cells were harvested and stained with anti-CD69 antibody. The means and SE of the proportions of CD69⁺ cells in a GFP⁺ gate of 5 independent experiments are indicated (middle). □, empty MIN; ■, MIN-CD8:CD3ϵ. To follow the kinetics, the cells were harvested at various days after CD8:CD3ϵ expression and the proportions of CD69⁺ and CD25⁺ cells in GFP⁺ gate were determined (right), SCID.adh cells transfected with empty MIN (○), SPA-1 (●), and Rap1A17 (■). Similar results were obtained in 2 independent experiments. (B) SCID.adh cells were infected with empty MIN or MIN containing C3G-F or FLAG-tagged Rap1E63, and the similar analysis described in panel A were performed. indicates FLAG-tagged Rap1E63; and ◀, endogenous Rap1.