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Fig. 6. R-Ras interacts with PLC ${epsilon}$ . (A) R-Ras co-immunoprecipitates with PLC ${epsilon}$ in Cos7 cells. Cos7 cells were transfected with Flag-PLC ${epsilon}$ and control vector (lane 1), R-Ras^WT (lane 2) or R-Ras38V (lane 3), lysed and PLC ${epsilon}$ immunoprecipiated with anti-Flag antibody. Association with R-Ras was determined by immunoblotting with anti-R-Ras antibody. (B-D) Active R-Ras interacts with the Ras-association domain, RA2, of PLC ${epsilon}$ . A pulldown assay was performed by incubating GST-RA2 with lysates from MCF10A cells expressing-R-Ras^38V (B). Alternatively, lysates were obtained from control MCF10A cells that were stimulated with 8-CTP-2Me-cAMP for 30 minutes (C) or stimulated by adhesion to fibronectin for 45 minutes to activate endogenous R-Ras. In B-D, lysates were incubated with GST (negative control), GST-Raf-RDB (a robust, positive control), and PLC ${epsilon}$ -RA2 domain for 2 hours at 4°C. Often endogenous R-Ras is noted as a doublet band, as seen here. (E) Constitutively active R-Ras activates PLC ${epsilon}$ activity. Cos7 cells were transiently transfected with PLC ${epsilon}$ and pCMV vector, pCMV-R-Ras^41A or pCMV-R-Ras^38V and a PLC activity assay was performed 48 hours post transfection. Data represent the means of two separate experiments each performed in duplicate. The minimum value of the y-axis is 100 cpm, which represents the background count. (F) Immunoblot for the PLC assay shown in E, demonstrating expression of Flag-PLC ${epsilon}$ as well as R-Ras^41A and R-Ras^38V in transfected, unlabelled Cos7 cells.

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