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Fig. 3. FGF2 stimulates recruitment of IQGAP1, N-WASP, Arp2/3 complex and FGFR1 to lamellipodia. (A) Low-density cultures of serum-starved MDBK cells were stimulated with FGF2, and lysed at various times thereafter. At each indicated time point, IQGAP1 was immunoprecipitated out of the lysates with monoclonal anti-IQGAP1, and the immunoprecipitates were analyzed by immunoblotting with antibodies to IQGAP1 (polyclonal), N-WASP, Arp3 and FGFR1. As a control for non-specific immunoprecipitation, the tau-1 monoclonal antibody to tau, which is not expressed in MDBK cells, was substituted for monoclonal anti-IQGAP1, 10 minutes after FGF2 stimulation. Note that the IQGAP1 immunoprecipitates contained time-dependent increases in the levels of coimmunoprecipitated N-WASP, Arp3 and FGFR1, and that none of the proteins assayed by western blotting were immunoprecipitated by anti-tau. (B) Glutathione-Sepharose 4B beads were loaded with a fusion protein of the FGFR1 cytoplasmic tail coupled to GST, or to GST alone, and the beads were then mixed with IQGAP1, N-WASP, or both, and finally immunoblotting was used to detect any IQGAP1 or N-WASP that may have bound to the beads. Immunoblotting demonstrated direct binding of IQGAP1 and indirect, IQGAP1-dependent association of N-WASP with GST-FGFR1 tail, but not with GST.
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