RASSF1A interacts with EWS. (A) Schematic of RASSF1A protein. The region (amino acids 1 to 210) used in a yeast two-hybrid screen for interacting proteins is indicated. (B) 293 cells were transfected with Myc-tagged RASSF1A and the green fluorescent protein (GFP)-tagged EWS zinc finger domain (amino acids 175 to 210). After 48 h, cells were lysed and α-myc-coupled agarose was used to immunoprecipitate RASSF1A. Lysates and immunoprecipitates were resolved by SDS-PAGE and immunoblotted with myc and green fluorescent protein antibodies as indicated. (C) HeLa cell lysates were immunoprecipitated with a RASSF1A-specific monoclonal antibody, G5. Lysates and immunoprecipitates were resolved by SDS-PAGE, transferred to polyvinylidene difluoride membrane, and immunoblotted with a polyclonal RASSF1A-specific antibody, 4169, and an EWS antibody as indicated. WB, Western blot; I.P., immunoprecipitate; WCL, whole-cell lysate; IgG, immunoglobulin G.