Fig. 3.

DGKι selectively inhibits RasGRP3. (A) HEK293 cells were transfected with RasGRPs along with the empty vector (–), Flag-DGKζ, or Flag-DGKι. After 24–48 h, active Ras (Ras-GTP) or Rap1 (Rap1-GTP) was detected by using GST-RBD pull-down assays. Total cell lysates were used to assess total Ras, Rap1, and DGK (n = 2–6 per experiment). (B) HEK293 cells were transfected with the indicated constructs. After 48 h, Rap1-GTP or Ras-GTP were affinity-precipitated and then detected by Western blotting. Data were analyzed by using scanning densitometry, and the results shown represent active Rap1 or active Ras normalized to total Rap1 or Ras in the lysates + SE (n = 3).