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Fig. 2. Mouse ARAP3 exhibits Arf GAP and Rho GAP activity in vitro. (A) FLAG-tagged ARAP3 was transiently expressed in 293T cells then immunoprecipitated with a FLAG monoclonal antibody. Arf GAP activity was assayed using bacterially expressed Arf5 pre-loaded with [ ${gamma}$ -³²P]GTP in the presence of 360 µM phosphatidic acid (PA), 45 µM PtdIns(4,5)P₂ (PIP2) and/or 1 µM PtdIns(3,4,5)P₃ (PIP3). GAP activity is expressed as the rate of GTP hydrolysis (per minute). (B) FLAG-tagged forms of ARAP3, AGAP1 and ACAP1 were expressed in 293T cells and assayed for Arf GAP activity towards bacterially expressed Arf1, Arf5 or Arf6 as described in A. Reaction buffers included 1 µM PtdIns(3,4,5)P₃ (ARAP3) or 45 µM PtdIns(4,5)P₂ plus 360 µM PA (AGAP1 and ACAP1). (C) A FLAG-tagged truncated ARAP3, (PH3-PH5), which includes the Rho GAP domain and flanking PH domains only, was expressed in 293T cells and immunopurified with a FLAG antibody. His-tagged RhoA, Rac1 or Cdc42 substrates were pre-loaded with [ ${gamma}$ -³²P]GTP then incubated for the indicated times in the presence (?) or absence ( ${Delta}$ ) of ARAP3 PH3-PH5. The results are expressed as the percentage [ ${gamma}$ -³²P]GTP that remains bound to the indicated Rho-family GTPases and therefore indicates the rate of GTP hydrolysis. The data are representative of two experiments.

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