Figure 1.

Upregulation of Rap1 activation in Cbl^−/− thymocytes. (A) Pull-down assay for activated Rap1 in thymocytes. Cell lysates from unstimulated or anti-CD3-stimulated thymocytes were incubated with 4 μg of either glutathione-S-transferase (GST) alone or a fusion of GST to the Rap1-binding domain of the Ral guanine-nucleotide dissociation stimulator (GST–RBD). The precipitate was blotted with anti-Rap1 (top panel) and the membrane was reprobed with anti-GST (bottom panel). (B,C) Thymocytes from Cbl^+/+ or Cbl^−/− mice were stimulated with anti-CD3 for 5 min (B) or for varying durations (C), followed by pull-down assays with GST or GST–RBD. Aliquots of cell lysates (CL) were blotted with anti-Rap1 or anti-Cbl as indicated.