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

FIG. 6.

FIG. 6.

RalGDS forms a complex with JIP1 in cells. (A) HA-Akt was transfected into COS-7 cells along with empty vector or vector expressing Flag-JIP1. JIP1 was immunoprecipitated (IP) with anti-Flag antibodies and then immunoblotted (IB) with anti-HA antibody to detect Akt (first row). Alternatively, Flag-JIP1 was immunoprecipitated and blotted with anti-Flag antibody (second row). Finally, lysates were immunoblotted with anti-HA antibody (third row) and anti-Flag antibody (fourth row). (B) Myc-RalGDS (Myc-GDS) was transfected into COS-7 cells along with either empty vector or vector expressing Flag-JIP1. JIP1 was immunoprecipitated with anti-Flag antibodies and then immunoblotted with anti-Myc antibody to detect RalGDS (first row). Alternatively, Flag-Jip1 was immunoprecipitated and blotted with anti-Flag antibody (second row). Samples were also blotted for Myc-RalGDS (third row) and Flag-JIP1 (fourth row). (C) Myc-RalGDS or empty vector was transfected into COS-7 cells along with Flag-JIP1. RalGDS was immunoprecipitated with anti-Myc antibodies and then immunoblotted with anti-Flag antibody to detect JIP1 (first row). Alternatively, Myc-RalGDS was immunoprecipitated and blotted with Myc antibody (second row). Finally, Flag-JIP and Myc-RalGDS were immunoblotted (third and fourth rows). (D) Flag-JIP1 was transfected into COS-7 cells along with empty vector or vector expressing Myc-RalGDS, Myc-RalGDSΔN (Myc-ΔN), or Myc-RalGDSΔC (Myc-ΔC). RalGDS or mutants were immunoprecipitated with anti-Myc antibodies, and the presence of JIP1 in the immunoprecipitates was assayed by immunoblotting with anti-Flag antibody (first row). Alternatively, Myc-RalGDS was immunoprecipitated and blotted with Myc antibody (second row). Expression levels of JIP and GDS in lysates were also evaluated (third and fourth rows). (E) Lysates of MCF10A cells stably expressing Myc-RalGDS were immunoprecipitated with either control mouse immunoglobulin G (IgG) or JIP1 antibodies and then samples were immunoblotted with anti-Myc (first row) or anti-JIP1 (second row) antibodies. Extracts were also blotted directly with anti-Myc antibodies to measure Myc-RalGDS levels (third row) and with anti-JIP1 antibodies to measure JIP1 levels (fourth row). Results are representative of experiments performed at least twice.