Cell lysates (∼1–2 mg of protein) were incubated 4 h at 4°C with either 2 μg of anti-EGFR (#225) mAb (endogenous EGFR), anti-FLAG mAb (Sigma-Aldrich) (FLAG-EGFR), and their respective preimmune control IgGs. Protein G-agarose beads (Invitrogen) were added and incubated at 4°C for an additional 60 min. Beads were washed then either resuspended and boiled in SDS sample buffer, or beads were incubated with purified His-GIV-CT (aa 1623–1870) overnight, washed, and then resuspended and boiled in SDS sample buffer. For immunoprecipitations involving ligand-activated EGFR, buffers used during all steps of the process were supplemented with 100 μM sodium orthovanadate.
For experiments investigating direct interaction between GIV and EGFR, recombinant EGFR kinase (Cell Signaling Technology) was used to phosphorylate GST-tagged EGFR C terminus (EGFR-T, aa 1064–1210) in vitro according to the manufacturer's protocol. In brief, equal aliquots of GST and GST-EGFR-T were incubated with 5 ng purified kinase in the presence of 200 μM ATP (Sigma-Aldrich) at room temperature for 60 min before their use in binding assays.
The in vitro binding assays using GST-fusion proteins were carried out as described previously (Ghosh et al., 2008 (link)). In brief, purified GST-fusion proteins (15–20 μg) or GST alone (30 μg) were immobilized on glutathione-Sepharose beads and resuspended in binding buffer supplemented with nucleotides (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 0.4% [vol/vol], NP-40, 10 mM MgCl2, 5 mM EDTA, 2 mM DTT, and protease inhibitor cocktail supplemented with either 30 μM GDP or 30 μM guanosine diphosphate [GDP], 30 μM AlCl3, and 10 mM NaF) (Ghosh et al., 2008 (link)). Thereafter, [35S]Met (GE Healthcare) -labeled GIV prepared using the TnT Quick Coupled Transcription/Translation System (Promega, Madison, WI) was added to the binding buffer, and binding was carried out overnight at 4°C with constant tumbling. The following day, the beads were washed (4.3 mM Na2HPO4, 1.4 mM KH2PO4, pH 7.4, 137 mM NaCl, 2.7 mM KCl, 0.1% [vol/vol] Tween 20, 10 mM MgCl2, 5 mM EDTA, and 2 mM DTT), and boiled in sample buffer for SDS-polyacrylamide gel electrophoresis. For Gαi3, the wash buffer was supplemented with GDP or GDP, AlCl3 and NaF during binding.
For experiments investigating direct interaction between GIV and EGFR, recombinant EGFR kinase (Cell Signaling Technology) was used to phosphorylate GST-tagged EGFR C terminus (EGFR-T, aa 1064–1210) in vitro according to the manufacturer's protocol. In brief, equal aliquots of GST and GST-EGFR-T were incubated with 5 ng purified kinase in the presence of 200 μM ATP (Sigma-Aldrich) at room temperature for 60 min before their use in binding assays.
The in vitro binding assays using GST-fusion proteins were carried out as described previously (Ghosh et al., 2008 (link)). In brief, purified GST-fusion proteins (15–20 μg) or GST alone (30 μg) were immobilized on glutathione-Sepharose beads and resuspended in binding buffer supplemented with nucleotides (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 0.4% [vol/vol], NP-40, 10 mM MgCl2, 5 mM EDTA, 2 mM DTT, and protease inhibitor cocktail supplemented with either 30 μM GDP or 30 μM guanosine diphosphate [GDP], 30 μM AlCl3, and 10 mM NaF) (Ghosh et al., 2008 (link)). Thereafter, [35S]Met (GE Healthcare) -labeled GIV prepared using the TnT Quick Coupled Transcription/Translation System (Promega, Madison, WI) was added to the binding buffer, and binding was carried out overnight at 4°C with constant tumbling. The following day, the beads were washed (4.3 mM Na2HPO4, 1.4 mM KH2PO4, pH 7.4, 137 mM NaCl, 2.7 mM KCl, 0.1% [vol/vol] Tween 20, 10 mM MgCl2, 5 mM EDTA, and 2 mM DTT), and boiled in sample buffer for SDS-polyacrylamide gel electrophoresis. For Gαi3, the wash buffer was supplemented with GDP or GDP, AlCl3 and NaF during binding.
