Small clusters of FGH-Ras and Lyn-FG formed in the inner leaflet triggered signal transduction. (A) Lyn-FG is likely functional because it exhibited self-phosphorylation (activation) similar to endogenous Lyn in RBL-2H3 cells after stimulation using anti-DNP IgE + DNP-BSA. Meanwhile, Lyn-FG oligomers induced by AP20187, an FKBP cross-linker, failed to activate Lyn-FG. Phosphorylation was detected by using the anti-pY418 Abs (taking the ratio of the anti-pY418 band versus the anti-Lyn band). (B) FGH-Ras is likely functional because it was pulled down, like endogenous Ras, by the Ras-binding domain (RBD) of the downstream molecule c-Raf kinase bound to polystyrene beads (detection with anti-Ras Abs) after EGF stimulation. FGH-Ras oligomerization by the addition of AP20187 induced FGH-Ras activation. AP21998 and methanol (MeOH) are negative controls. (C and D) Histograms showing the distributions of the signal intensities of individual fluorescence spots of Ly-FG (C) and FGH-Ras (D) before (top) and after (bottom) the addition of AP20187. Based on these histograms, we concluded that each Lyn-FG cluster and FGH-Ras cluster contained an average of approximately three Lyn-FG and FGH-Ras molecules, respectively.