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Figure 2. The NH2-terminal domain of N-WASP inhibits binding of Arp2/3 complex to the COOH-terminal VCA segment in a Cdc42- and PI(4,5)P2-regulated manner. (A) Purified protein preparations of the hexahistidine-tagged (HT) WG and WGP fragments and GST-VCA used in this study are shown on a 5–15% SDS–polyacrylamide gel. (B) Binding of GST-VCA or GST-fused full-length N-WASP (0.5 μM each) to Arp2/3 complex (0.1 μM) was assayed by incubating the components in solution and capturing the GST fusion protein on glutathione-Sepharose beads. The amount of Arp2/3 bound was determined by analyzing 50% of the input and 37.5% of the pulled down material on an α-Arp2 immunoblot. (C) Binding of GST-VCA (0.2 μM) to Arp2/3 (0.1 μM) was tested (as in B) in the presence of the indicated concentrations of HT-WG or HT-WGP. Immunoblotting was used to assay the amount of GST-VCA (α-GST), Arp2/3 (α-Arp3 and α-p34Arc), and HT-WG or HT-WGP (α-pentaHis) precipitated on the glutathione-Sepharose beads. (D) The ability of HT-WG (2 μM) to inhibit the binding of Arp2/3 (0.1 μM) to GST-VCA (0.2 μM) was tested in the presence of GTPγS-loaded Cdc42 (5 μM) or PI(4,5)P2-containing vesicles (100 μM; PI(4,5)P2/PI/PC = 10:45:45). (E) GST-Cdc42 beads, loaded with either GTPγS or GDP, were tested for their abilities to bind to Arp2/3 (1 μM) in the presence of N-WASP (1 μM) and PI(4,5)P2-containing vesicles (100 μM). Arp2/3 pulled down by GST-Cdc42 beads in the absence of any N-WASP represents nonspecific, background binding under the conditions of the assay. For the α-Arp2 immunoblot, 7.5% of the input, 10% of the material pulled down on the GST-Cdc42 beads, and (as a control for comparison) 10% of the material pulled down by GST-VCA (1 μM) on glutathione- Sepharose beads was loaded on the gel. Image published in: Rohatgi R et al. (2000) © 2000 The Rockefeller University Press. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike license Larger Image Printer Friendly View

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