FIGURE 6.

Src promotes the integration of PIPKIγi2 and PI3K into a complex. A, PIPKIγi2 or Src was immunoprecipitated from MDA-MB-231 cells co-expressing PIPKIγi2 and Src. The coimmunoprecipitation of PI3K enzyme into the immunocomplex was examined by immunoblotting using the antibody specific for PI3K subunits (e.g. p85 and p110α). B, MDA-MB-231 cells expressing PIPKIγi2 were incubated in the suspension condition or stimulated with EGF and ECM for the indicated time periods. PIPKIγi2 was immunoprecipitated, and coimmunoprecipitation of Src and PI3K into the immunocomplex was examined by immunoblotting. C, MDA-MB-231 cells expressing PIPKIγi1 or PIPKIγi2 were stimulated with EGF and ECM protein in the suspension condition as described above. Then PIPKIγi1 or PIPKIγi2 was immunoprecipitated to examine the coimmunoprecipitation of Src and PI3K into the immunocomplex. D, PIPKIγi2 was immunoprecipitated from MDA-MB-231 cells transfected with siRNA for the knockdown of Src. Cells were stimulated as described above, and the coimmunoprecipitation of PI3K with PIPKIγi2 in the immunocomplex was examined by immunoblotting. E, endogenous Src was immunoprecipitated from HEK293 cells transfected with siRNA for PIPKIγ or PIPKIγi2 knockdown. The coimmunoprecipitation of PI3K with Src was examined by immunoblotting. F, HEK293 cells were co-transfected with Src and p85. Src was immunoprecipitated, and coimmunoprecipitation of p85 was examined by immunoblotting. Reciprocally, coimmunoprecipitation of Src with p85 was examined.