FIGURE 1.

IQGAP1 is a novel SKAP interacting partner at leading edge of migrating cells. A, SKAP forms a novel complex with IQGAP in interphase cells. Aliquots of MDA-MB-231 cells were collected by centrifugation and extracted with Triton X-100-containing buffer as described under “Materials and Methods.” Clarified cell lysates were incubated with Sepharose beads affinity matrix covalently coupled with SKAP antibody and control rabbit IgG, respectively, as described under ”Materials and Methods.“ The beads were washed successively with PBS before elution with 0.2 m glycine, pH 2.3. All samples were separated by SDS-gel electrophoresis. B, SKAP immunoprecipitates from MDA-MB-231 cells in interphase were immunoblotted with antibodies against SKAP, IQGAP, MCAK, and EB1. Note that SKAP immunoprecipitation brought down IQGAP1 and EB1 but not MCAK. C, IQGAP immunoprecipitates from MDA-MB-231 cells in interphase were immunoblotted with antibodies against SKAP, IQGAP, MCAK, and Cdc42. Note that IQGAP1 immunoprecipitation brought down IQGAP1 and Cdc42 but not MCAK. D, MDA-MB-231 cells were labeled with antibodies against SKAP (red), tubulin (green), and IQGAP1 (blue). Scale bar, 10 μm. Note that the membrane ruffle-like localization of SKAP is superimposed onto that of the IQGAP1 signal and the three channels are merged, although the SKAP signal at membrane ruffles is less intense compared with IQGAP1 (d′, arrows). Bar, 10 μm.