Figure 1

Rasd1 and Ear2 interact in vitro and in living cells. (A) In vitro binding of GST-Ear2 and HisHA-Rasd1. COS-7 cells were transfected with plasmids expressing the indicated constructs. Cell lysates containing HisHA-Rasd1 was incubated with immobilized GST-Ear2 or GST, and specifically bound HisHA-Rasd1 was eluted by heating at 95°C for 10 minutes, and detected by Western blotting with Anti-HA antibody. (B) HisHA-Rasd1 and GST-Ear2 interact in intact mammalian cells. pHisHA-Rasd1 was co-transfected with plasmids expressing either GST-Ear2 or GST into COS-7 cells. GST and GST-Ear2 were captured from the cell lysates by GSH-linked beads and HisHA-Rasd1 bound to the beads were detected as above. (C) COS-7 cells were co-transfected with plasmids expressing HisHA-Rasd1 and GST-Ear2 or GST. Immunoprecipitations were performed with anti-GST antibody. Co-immunoprecipitated HisHA-Rasd1 was detected by Anti-HA antibody. (D and E) Rasd1 and Ear2 form a physiological complex in living cells. Endogenous Rasd1-Ear2 complexes were detected by immunoprecipitating Ear2 from HEK293T cell lysates (D) or mouse brain lysates (E) with goat polyclonal IgG anti-Ear2 antibody, and probing for Rasd1 (D and E, lanes 1). A non-relevant goat polyclonal IgG antibody was used as a negative control (D and E, lanes 2). IP and IB denote immunoprecipitation and immunoblot, respectively.