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. 2008 Feb 19;28(9):2851–2859. doi: 10.1128/MCB.01917-07

FIG. 7.

FIG. 7.

RalGDS forms a complex with Akt. (A) HA-Akt was transfected into COS-7 cells along with Myc-RalGDS or Myc-RalGDSΔN. Myc-RalGDS or Myc-RalGDSΔN was immunoprecipitated (IP) with anti-Myc antibodies and then immunoblotted (IB) with anti-HA antibody to detect Akt (first row) and immunoblotted with anti-Myc antibodies to detect Myc-GDS (second row). HA-Akt and Myc-GDS expression levels in lysates are shown in the third and fourth rows. (B) HA-Akt was transfected into COS-7 cells along with Myc-RalGDS, Flag-JIP1, and empty vector. RalGDS was immunoprecipitated with anti-Myc antibodies and then immunoblotted with either anti-HA antibody to detect Akt (first row), anti-Flag antibody to detect JIP1(second row), or anti-Myc antibody to detect GDS (third row). HA-Akt, Flag-JIP1, and Myc-GDS expression levels are shown in fourth, fifth, and sixth rows. (C) Top: Extracts of COS-7 cells (Con) or COS-7 cells constitutively expressing shRNAi against JIP1 (shRNA) were immunoblotted with anti-JIP1 antibodies or total Akt antibodies. Bottom: COS-7 cells or COS-7 cells expressing JIP1 shRNA were transfected with Glu-RalGDS, and either empty vector or vector expressing Myc-Akt. Myc-Akt was immunoprecipitated, and the sample was immunoblotted with anti-Glu-GDS antibodies (first row) or My-AKT antibodies (second row). Lysates were also blotted with anti-Glu-GDS antibodies (third row) or anti-Myc antibodies (fourth row). (D) COS-7 cells or COS-7 cells expressing JIP1 shRNA were serum starved and then stimulated with EGF for 5 min. Lysates were immunoblotted with pAKT T308-specific antibodies. (E) HA-Akt or Flag-JIP1 was transfected into COS-7 cells along with Myc-RalGDS. Cells were serum starved for 6 h and then stimulated with EGF (5 ng/ml) for 5 min. Cell lysates were immunoprecipitated with anti-Myc antibody and then immunoblotted with anti-HA antibody to detect Akt and with anti-Flag antibody to detect JIP1. Samples were also blotted with HA, Myc, or Flag antibodies to detect expression levels of proteins in cells. Samples were also immunoblotted with pAKT antibodies to confirm EGF stimulation was effective. Data are representative of experiments performed at least twice.