Antibodies and constructs
Affinity-purified rabbit anti-APPL1 and anti-Gαs immunoglobulin G (IgG) were gifts from Joseph Testa (Fox Chase Cancer Center, Philadelphia, PA) and Allen M. Spiegel (Albert Einstein College of Medicine, New York, NY). Mouse anti-Rab5 monoclonal antibody (mAb; clone 4F11-D9-C4) was a gift from Angela Wandinger-Ness (University of New Mexico, Albuquerque, NM). Rabbit antibodies against Girdin/GIV, pan-Gβ, Grb2, Hrs, and EGFR (sc-03) were purchased from Santa Cruz Biotechnology, Gαi3 and Gαs from Calbiochem (La Jolla, CA), and those against phospho–histone H3 (P-H3, Ser-10) from Millipore (Billerica, MA). Rabbit antibodies against pY1045-EGFR, pY1068-EGFR, pAkt (Ser-473), total Akt, pERK1/2, and EEA1 were purchased from Cell Signaling Technology (Beverly, MA).
Mouse mAbs were purchased as follows: GFP (Clontech, Mountain View, CA), LAMP-2 (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), EGFR #528 (Calbiochem), EEA1 and BrdU (clone B44) for immunofluorescence (BD Biosciences), actin, tubulin, and BrdU for flow cytometry (Sigma-Aldrich, St. Louis, MO), and c-Cbl (BD Biosciences, San Diego, CA). Control mouse IgGs used for immunoprecipitation were purchased from Cell Signaling Technology.
For immunofluorescence, highly cross-absorbed Alexa Fluor 594 or 488 F(ab′)2 fragment of goat anti-mouse or anti-rabbit IgG (H+L) were purchased from Invitrogen (Carlsbad, CA). For immunoblotting, goat anti-mouse IgG (H+L) IRDye 800 and goat anti-rabbit Alexa Fluor 680 F(ab′)2 were purchased from Rockland Immunochemicals (Gilbertsville, PA) and Invitrogen, respectively.
For immunoelectron microscopy, sheep anti-EGFR IgG was purchased from Fitzgerald Industries International (Acton, MA), rabbit anti-goat IgG (used as a bridge) from Nordic Immunological Laboratories (Eindhoven, Netherlands), and 10-nm gold-conjugated goat anti-rabbit IgG from Amersham Biosciences (GE Healthcare, Piscataway, NJ).
Gαs-GFP in pCDNA3.1 was a gift from Mark Rasenick (University of Illinois at Chicago, Chicago, IL). Human WT and constitutively active Q227L (QL) Gαs (long and short isoforms) in pCDNA3.1 were purchased from Guthrie cDNA Resource Center (Missouri University of Science and Technology, Rolla, MO). pCDNA3.1 Gαs-G226A (GA) mutant was made using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, Santa Clara, CA). siRNA-resistant Gαs WT, QL, and GA mutant constructs were made by introducing silent substitutions in the Gαs cDNA within the region of homology to the siRNA Gαs oligo (Zheng et al., 2004
). N-terminally tagged FLAG-Rab5-QL was subcloned into p3XFLAG-CMV (Sigma-Aldrich). Human GIV was N-terminally tagged with CFP and cloned into the pAP4 vector. Gαs-WT (human, short) was cloned into pGEX-KG and purified as described previously (Ghosh et al., 2008
). All primers are available upon request.
Cell culture, transfection, and EGF stimulation
HeLa and Cos7 cells (American Type Culture Collection, Manassas, VA) were grown in DME (Invitrogen) supplemented with 10% FBS (Hyclone, Logan, UT) and penicillin–streptomycin–glutamine (Invitrogen).
For transfection of siRNA oligos, HeLa cells were seeded (5.5 × 104 cells/35 mm dish or 5.5 × 105 cells/10 cm dish), and 24 h later they were transfected with 20 nM final siRNA for 14 h using Oligofectamine (Invitrogen) according to the manufacturer's instructions.
For reversal of the effects of Gαs depletion, 1 × 105 HeLa cells were seeded/well (six-well dish) or 5.5 × 105/10 cm dish, transfected with 20 nM siRNA oligo using Oligofectamine overnight as described, and then transfected with 2 μg/well or 10 μg/dish plasmid DNA (pCDNA3.1 alone or pCDNA3.1 siRNA-resistant Gαs-WT, QL, or GA mutants) for 8 h using FuGENE (Roche, Indianapolis, IN) or TransIT-LT1 (Mirus Bio, Madison, WI) according to the manufacturer's instructions.
For expression of FLAG-Rab5-QL and CFP-GIV, HeLa cells were transfected first with control and Gαs siRNA for 14 h using Oligofectamine and then cotransfected with 1 μg of FLAG-Rab5-QL and 2 μg of CFP-GIV for 8 h using TransIT-LT1.
For expression of GFP or Gαs-GFP, 10-cm dishes of Cos7 cells were transfected with 2 μg GFP or 10 μg Gαs-GFP for 14 h using TransIT-LT1.
For EGF stimulation experiments, 48 h after siRNA transfection the serum concentration was reduced from 10 to 0.2% overnight before stimulation with 50 nM EGF (mouse submaxillary gland; Invitrogen), 300 ng/ml Texas-red EGF (Invitrogen), or 300 ng/ml Alexa 488 EGF (Invitrogen) in DME alone.
Immunofluorescence and immunoelectron microscopy
For immunofluorescence analysis, cells grown on coverslips were fixed in 3% paraformaldehyde (PFA) for 30–60 min at room temperature (RT), quenched (50 mM NH4Cl2), blocked (10% normal goat serum), permeabilized (0.1% Triton X-100 [TX-100] in phosphate-buffered saline [PBS]), incubated in primary antibodies (1 h at RT) or overnight at 4°C (for phosphospecific IgGs) and then secondary IgGs (1 h at RT), and mounted on slides in 1% propyl-gallate (Sigma-Aldrich) in 1:1 glycerol:PBS. Antibody dilutions were as follows: EEA1, 1:200; pY1068-EGFR, 1:150; GIV, 1:180; APPL1, 1:200; phospho–histone H3, 1:150; EGFR (#528), 1:300; EEA1, 1:200; BrdU, 1:5; LAMP-2, 1:400; goat anti-mouse or anti-rabbit Alexa 488 or Alexa 594, 1:500. 4′,6-Diamidino-2-phenylindole (DAPI) (Invitrogen) was used at 1:3000. Confocal imaging was carried out on an inverted IX81 microscope (Olympus, Tokyo, Japan) equipped with 405-, 488-, 560-, and 640-nm laser lines, UltraView Vox Spinning Disk Confocal (PerkinElmer, Waltham, MA), a 60× oil (differential interference contrast) lens, an electron-multiplying charge-coupled device (CCD) Hamamatsu 14-bit camera (Hamamatsu, Hamamatsu, Japan), and Volocity software (PerkinElmer; University of California, San Diego, School of Medicine Light Microscopy Facility).
For immunoelectron microscopic studies, cells were fixed 4 h in 4% PFA, followed by 12 h in 1% PFA in 0.1 M phosphate buffer, pelleted in 10% gelatin, cryoprotected in sucrose, and snap frozen in liquid nitrogen. Ultrathin cryosections (70–80 nm) were cut as previously described (Zheng et al., 2004
). For immunogold labeling of EGFR, sections were incubated sequentially with sheep anti-EGFR IgG (2 h), rabbit anti-goat IgG (bridging antibody; 1 h), and goat anti-rabbit IgG-gold conjugates (1 h) and then contrasted (10 min in 0.4% uranyl acetate and 1.8% methyl cellulose on ice). Imaging was carried out using a JEOL 1200 EX II electron microscope equipped with an Orius CCD Gatan camera and Gatan digital micrograph software (Gatan, Pleasanton, CA; University of California, San Diego, Cellular and Molecular Medicine Electron Microscopy Facility).
Whole-cell lysis and immunoblotting
Cells were harvested, suspended in 2.5× Laemmli sample buffer, and boiled for 15 min. Samples were separated by 10% SDS–PAGE or 15% SDS–PAGE (for P-H3 analysis) and transferred to PVDF-FL membranes (Millipore). Membranes were blocked (5% BSA, 0.1% Tween-20 in PBS) and incubated with primary antibodies (4°C overnight) and then with secondary antibodies (30 min at RT). Bands were imaged and quantified by two-color detection with the Odyssey infrared imaging system (Li-Cor Biosciences, Lincoln, NE). Antibody dilutions were as follows: pY1068- and pY1045-EGFR, pAkt, tAkt, Girdin/GIV, tERK1/2, Hrs, and c-Cbl, 1:250; Gαs, Gαi3, tEGFR, EEA1, Rab5, and APPL1, 1:500; Grb2, pERK1/2, actin, GFP, P-H3, and tubulin, 1:1000 to 1:2000; and goat anti-rabbit Alexa Fluor 680 and goat anti-mouse IRDye 800 F(ab′)2, 1:15,000.
To immunoprecipitate endogenous EGFR, control or Gαs deleted cells were harvested, lysed in buffer A (0.4% TX-100, 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.2, 5 mM Mg acetate, 125 mM K acetate, 1 mM dithiothreitol [DTT], and 20 mM N-ethylmaleimide supplemented with Complete Protease Inhibitor cocktail [Roche] and Phosphatase Inhibitor Cocktail 2 [Sigma-Aldrich] inhibitors), incubated on ice (1 h) with vortexing every 10 min, and cleared by centrifugation (10,000 × g for 10 min). Cell lysates were incubated overnight at 4°C with either control or anti-EGFR mAb #528. Protein G–Sepharose beads (GE Health Sciences) were added and incubated an additional 60 min. Beads were washed, suspended, and boiled in Laemmli sample buffer, and bound proteins were analyzed by immunoblotting.
To immunoprecipitate GFP or Gαs-GFP, Cos7 cells were transfected for 48 h with GαsGFP or GFP alone, harvested, suspended in buffer A supplemented with 1 mM DTT, 10 mM MgCl2, and 30 μM GDP ± 10 mM NaF and 10 μM AlCl3, incubated on ice, and cleared by centrifugation as described. Cell lysates were incubated overnight at 4°C with anti-GFP mAb. Bound proteins were recovered and analyzed as described.
Cell proliferation assays
For P-H3 analyses, whole-cell lysates were prepared as described and analyzed for P-H3 by immunoblotting (1:2000) or by immunofluorescence (1:150) as described previously (Lehtonen et al., 2008
; Ghosh et al., 2010
For BrdU (Sigma-Aldrich) incorporation experiments, control or Gαs-depleted HeLa cells were incubated in 10 μM BrdU for 30 min at 37°C in DME supplemented with 10% FBS. Immunofluorescence and flow cytometry analysis of incorporated BrdU were carried out according to the manufacturer's instructions (Sigma-Aldrich). Briefly, cells were trypsinized, suspended in PBS, and fixed in 100% ethanol (30 min, RT). Samples were incubated in 2 N HCl (20 min, RT), followed by 0.1 M sodium borate (5 min) and mouse anti-BrDU (1:8 dilution; BD Biosciences, San Diego, CA) for 30 min, followed by goat anti-mouse Alexa Fluor 488 for 30 min. RNA was digested, and DNA content was stained by incubation in 100 μg/ml RNase and 5 μg/ml propidium iodide (PI) for 20 min at 37°C. Samples were filtered (10-μm Nitex nylon mesh; Sefar America, Depew, NY), loaded onto an LSRII flow cytometer (BD Biosciences), and analyzed using FlowJo software (TreeStar, Ashland, OR). Cells untreated or exposed only to anti-BrdU antibody or PI were used for background measurements.
Preparation of membrane and cytosolic fractions
Cells were harvested and suspended in 3 mM imidazole buffer in 250 mM sucrose, with protease and phosphatase inhibitors, and homogenized by passage (30×) through a 22-gauge needle as previously described (Felberbaum-Corti et al., 2005
). Postnuclear supernatants (prepared by centrifugation of homogenates at 1200 × g
for 10 min) were centrifuged for 1 h at 120,000 × g
, the cytosolic fraction (120,000 × g
supernatant) was collected, and the membrane fraction (120,000 × g
pellet) was resuspended in one-half the volume of homogenization buffer. Equal-volume samples of cytosolic and membrane fractions were resuspended in 2× sample buffer and analyzed by immunoblotting.
In vitro protein-binding assays
A total of 20 μg of purified GST-Gαs or GST alone was immobilized on glutathione–Sepharose (GE Healthcare) in buffer B (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, protease and phosphatase inhibitors, 30 μM GDP, 12 mM DTT, ± 30 μM AlCl3
, and 10 mM NaF) as described (Garcia-Marcos et al., 2009
). Immobilized GST-Gαs or GST was incubated overnight at 4°C with purified histidine-tagged GIV-CT (1623-1870) or HeLa or Cos7 cell lysates prepared in buffer A as for immunoprecipitation. Beads were boiled in 2× sample buffer and analyzed by immunoblotting.
Statistical and image analysis
Each experiment presented in the figures is representative of at least three independent experiments. All averages, SEMs, and significance p values (t test) were calculated and graphed using Excel (Microsoft, Redmond, WA). Quantification of IF images was carried out using Volocity software. All images were processed and figures assembled using Photoshop software (Adobe, San Jose, CA).