Unless otherwise indicated, all reagents were of analytical grade and obtained from Sigma-Aldrich (St. Louis, MO). Cell culture media were purchased from Invitrogen (Carlsbad, CA). Activated EGFR kinase and phospho-tyrosine monoclonal antibody (mAb) were purchased from Cell Signaling Technology (Danvers, MA). Silencer Negative Control #1 small interfering RNA (siRNA) and siRNA Gαi3 were purchased from Ambion (Austin, TX) and Santa Cruz Biotechnology (Santa Cruz, CA), respectively. Streptavidin-horseradish peroxidase (HRP), biotinylated EGF, and mouse submaxillary EGF were purchased from Invitrogen, and Rhodamine Red X-anti-HRP was from Jackson ImmunoResearch Laboratories (West Grove, PA). Antibodies against GIV that were used in this work include rabbit serum and affinity-purified anti-GIV coiled-coil immunoglobulin (Ig)G (GIV-ccAb) raised against the coiled-coil domain of GIV (Le-Niculescu et al., 2005 (link); Ghosh et al., 2008 (link)), and affinity-purified anti-Girdin C terminus (GIV-CTAb) raised against the last 19 aa of GIV's C terminus (IBL America, Minneapolis, MN). To visualize total EGFR by immunofluorescence, mAb #225 raised against the ectodomain (gift from Gordon Gill, University of California San Diego [UCSD], La Jolla, CA; Gill et al., 1984 (link)) or polyclonal antibody (pAb) anti-EGFR against the C-terminus of EGFR (Cell Signaling Technology). Polyclonal phosphosite-specific EGFR antibodies (pY992, pY1045, and pY1068; Cell Signaling Technology), mAb pY845 (Millipore, Billerica, MA), and total EGFR (tEGFR) antibodies (Cell Signaling Technology and Santa Cruz Biotechnology) were used for immunoblotting. Rabbit polyclonal antibodies (pAb) against Gαi3 (Calbiochem, San Diego, CA) for immunofluorescence, Gαi3 (M-14, Santa Cruz Biotechnology) for immunoblotting, STAT5b and Grb2 (Santa Cruz Biotechnology), phospho-Y527 Src, PLCγ1, phospho-Y783 PLCγ1, phospho-S473 Akt, and phospho-ERK1/2 (Cell Signaling Technology), were obtained commercially. Mouse monoclonal antibodies (mAb) against phospho-Y845 EGFR and phospho-Y694/Y699-STAT5b (Millipore), Akt and EEA1 (BD Biosciences, San Jose, CA), c-Src (Santa Cruz Biotechnology), ERK1/2 (Cell Signaling Technology), and tubulin (Sigma-Aldrich) were obtained from commercial sources. Anti-mouse and anti-rabbit Alexa-594– and Alexa-488–coupled goat secondary antibodies for immunofluorescence (IF) were purchased from Invitrogen. Goat anti-rabbit and goat anti-mouse Alexa Fluor 680 or IRDye 800 F(ab`)2 for immunoblotting were from LI-COR Biosciences (Lincoln, NE). Control mouse and rabbit IgGs for immunoprecipitations were purchased from Bio-Rad Laboratories (Hercules, CA) and Sigma-Aldrich, respectively.