Fig. 3. The interaction of PLC-1 with dynamin-1 is essential for its GEF activity for dynamin-1. (A) PC12 cells stably transfected with various PLC-1 constructs were immunoprecipitated with anti-FLAG antibody. The cells were then immunoblotted with anti-dynamin antibody. (B) The stably transfected PC12 cells were metabolically labeled in phosphate-free DMEM with 0.5 mCi/ml [³²P]H₃PO₄ for 4 hours at 37°C and treated with EGF for the indicated times. The bound guanine nucleotides to dynamin-1 were analyzed by TLC. The ratio of GTP was calculated as GTP/(GTP+GDP). (C) PC12 cells overexpressing PLC-1^WT were transfected with HA-tagged dynamin-1^WT and dynamin-1^PRD constructs. Immunoprecipitation with anti-HA antibody was followed by immunoblotting with anti-PLC-1 and anti-HA antibody. (D) PC12 cells overexpressing PLC-1^WT were transfected with HA-dynamin-1 and its mutant. The bound guanine nucleotides to dynamin-1 were analyzed by TLC. The ratio of GTP was calculated as GTP/(GTP+GDP). Stars indicate a significant difference compared with the control.