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. 2011 Jul 28;286(37):32404–32415. doi: 10.1074/jbc.M111.275685

FIGURE 4.

FIGURE 4.

SHP-1 interacts with GIV in vivo. A, GIV co-immunoprecipitates with SHP-1, but not SHP-2. Equal aliquots of COS7 lysates (left) were incubated with either rabbit preimmune (first lane) or anti-SHP-1 (second lane) or anti-SHP-2 (third lane) IgGs and protein A-agarose beads. Immune complexes (25) were analyzed for GIV, SHP-1, and SHP-2 by immunoblotting (IB). SHP-1 and SHP-2 were immunoprecipitated efficiently and specifically. GIV co-immunoprecipitated with SHP-1 (second lane) but not SHP-2 (third lane) or control IgG (first lane). B, GIV, but not Gαi3, interacts constitutively with SHP-1. COS7 cells were serum-starved (−) and subsequently stimulated with 50 nm EGF for 10 min (+). Equal aliquots of lysates (bottom) were incubated with either rabbit preimmune (lane 1) or anti-SHP-1 (lanes 2 and 3) IgGs and protein A-agarose beads. Immune complexes (top) were analyzed for GIV, SHP-1, and Gαi3 by immunoblotting. SHP-1 was immunoprecipitated efficiently and specifically. GIV, but not Gαi3, co-immunoprecipitated with SHP-1 from both starved and EGF-stimulated cells (lanes 2 and 3). C, GIV GEF motif is not required for the GIV-SHP-1 interaction. COS7 cells were transfected with vector alone (lane 1) or FLAG-tagged wild-type GIV (GIV-WT; lane 2), or the GEF-deficient F1685A (FA) mutant (GIV-FA; lane 3). Equal aliquots of lysates (bottom) were incubated with anti-SHP-1 IgGs and protein A-agarose beads. Immune complexes were analyzed for FLAG (GIV-FLAG) and SHP-1 by immunoblotting.