C2A domain of Slp1 is sufficient for binding of Slp1 to Rap1GAP2. (A) Pull-down of transfected Rap1GAP2 with GST-Slp1 mutants. Lysates of HeLa cells overexpressing FLAG-tagged Rap1GAP2 (RG2-FLAG) were subjected to pull-down experiments using equal amounts of GST as control and GST fusion proteins of C2A (amino acids 292-393 of Slp1), C2B (amino acids 433-580 of Slp1), and C2AB (amino acids 292-580 of Slp1). (Top panel) The precipitates were analyzed for the presence of bound Rap1GAP2 by immunoblot with anti-FLAG antibody. (Bottom panel) Expression levels of Rap1GAP2 (total RG2-FLAG, 2% input). (B) Coimmunoprecipitation of transfected Rap1GAP2 and Slp1-C2A. HeLa cells were transfected with FLAG-tagged Rap1GAP2, myc-tagged C2A domain of Slp1, or FLAG-tagged Rap1GAP2 together with myc-tagged Slp1-C2A. After lysis, Rap1GAP2 was precipitated with anti-FLAG antibody. (Top panel) The precipitates were analyzed for the presence of bound Slp1-C2A by immunoblot using anti-myc antibody. (Bottom 2 panels) Expression levels of Slp1-C2A (total C2A-myc, 2% input) and Rap1GAP2 (total RG2-FLAG, 2% input).