Figure 3. Rit effector loop mutants exhibit differential binding to RGL3-RBD.

PC6 cells were transiently transfected with the indicated expression vectors, and the cells starved with serum-free DMEM for 5 h prior to the preparation of whole cell lysates. Cell lysates (200 μg) were incubated at 4 °C for 1 h with GST-RGL3-RBD pre-bound to glutathione beads. The RGL3-RBD complexed affinity beads were pelleted and washed extensively, and the amount of bound Rit protein in the pellet fraction was determined by anti-Flag immunoblotting. The expression of Rit present in each lysate was also determined by immunoblot analysis. Blots are representative of three independent trials.