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Fig. 2. The SH3 domain of PLC- ${gamma}$ 1 functions as a guanine nucleotide exchange factor (GEF) for dynamin-1 in vivo. (A) PC12 cells stably transfected with PLC- ${gamma}$ 1 constructs under a tet-off system were induced to express wild-type PLC- ${gamma}$ 1. Cells were then treated with 10 ng/ml EGF for the indicated times. Co-immunoprecipitated PLC- ${gamma}$ 1 was analyzed by immunoblotting with anti-PLC- ${gamma}$ 1 antibody. (B) PC12 cells stably transfected with wild-type PLC- ${gamma}$ 1 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 5 minutes. Dynamin-1 was immunoprecipitated with anti-dynamin-1 antibody, and bound guanine nucleotides were eluted with 1M KH₂PO₄ and separated on TLC plates. The ratio of GTP was calculated as GTP/(GTP+GDP) (Jeong et al., 2001; Rosen et al., 1994). (C) PC12 cells transfected with specific siRNA for PLC ${gamma}$ 1 (described in Materials and Methods) were stimulated with EGF for the indicated times. The expression of PLC-ß1, PLC-ß3 and PLC- ${gamma}$ 2 were analyzed by immunoblotting with anti-PLC-ß1, PLC-ß3 and PLC- ${gamma}$ 2 antibodies. (D) PC12 cells were transfected with siRNA for PLC- ${gamma}$ 1 metabolically labeled in phosphate-free DMEM with 0.5 mCi/ml [³²P]H₃PO₄ for 4 hour sat 37°C and treated with EGF for 5 minutes. 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.

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