mercaptoethanol) for 5 min. For immunoprecipitation, transfect- ed HEK293T lysates containing 200 mg protein were incubated with either a rabbit polyclonal anti-Hic-5 antibody (4914; Cell Signaling Technology Inc., Beverly, MA), mouse monoclonal anti-EGFR antibody (GR13; EMD Biosciences, Philadelphia, PA) and Protein A/G+ agarose beads (2003; Santa Cruz Biotechnology, Santa Cruz, CA) or HA-affinity matrix (11815016001; Roche Diagnostics Corp., Indianapolis, IN) overnight at 4uC with tumbling. Immune complexes were washed five times in cold lysis buffer, resuspended in 2 6 loading buffer and boiled for 5 min. The proteins were resolved by SDS-PAGE and transferred to nitrocellulose membranes (Protran BA85; Whatman, Sanford, MA). Immunoprecipitation from HeLa and CFPAC-1 lysates containing 2000 mg protein were incubated with rabbit polycolonal anti-Cbl-c antibody (Rockland, Info) and Protein A/G + agarose beads overnight at 4uC with tumbling. Whole IPs were run over spin columns (Thermo Scientific, Rockford, IL) and washed with 500 mls of lysis buffer a total of three times. Proteins were eluted with 30 ml Non-Reducing Lane Marker Sample Buffer (Thermo Scientific, Rockford, IL). Pull downs of GST tagged proteins were performed by incubating 300 mg of cell lysate with 35 ml of glutathione sepharose beads (GE Healthcare, Pascataway, NJ) overnight at 4uC with tumbling. Pull downs were washed five times with 750 ml of lysis buffer and the precipitated proteins were resuspended in loading buffer and boiled for 5 min. Membranes were probed with either mouse monoclonal anti- Hic-5 antibody (611164; BD Transduction Laboratories, Frank- lin Lakes, NJ), Horseradish peroxidase linked mouse monoclonal anti-HA (3F10; Roche Diagnostics Corp., Indianapolis, IN), rabbit monoclonal anti-GFP (SC8334; Santa Cruz Biotechnolo- gy, Santa Cruz, CA), rabbit polyclonal anti-phospho-Src (2105; Cell Signaling, Danvers, MA), rabbit polyclonal anti-EGFR (2232; Cell Signaling, Danvers, MA), or anti-phosphotyrosine 4G10 Platinum (0501050; EMD Millipore Corp, Billerica, MA). Horseradish peroxidase linked donkey anti-rabbit IgG (NA934V; GE Healthcare, Piscataway, NJ), Horseradish peroxidase linked donkey anti-mouse IgG (NA931: GE Healthcare, Piscataway, NJ), or rabbit anti-goat (SC2768; Santa Cruz Biotechnology, Santa Cruz, CA) immunoglobulin was used with SuperSignal (Pierce Biotechnology Inc., Rockford, IL) to visualize the blots. Confocal Microscopy HeLa cells (26105/well) were grown in a 6-well tissue culture dish in DMEM supplemented with 10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin sulfate. 24 h after plating, cells were transiently transfected with YFP-Cbl-c and CFP-Hic-5 plasmids individually or together (2.0 ug DNA per well) using Lipofectamine-2000 reagent (Invitrogen, Carlsbad, CA) according to the manufacturer recommendations. 48 h post-transfection, cells were trypsinized and 26104cells were seeded on each well of 4-chambered coverglass slides (Lab-Tek, Scotts Valley, CA). Cells were rinsed twice with PBS and fixed in 4% paraformaldehyde for 10 min at room temperature. HeLa cells expressing YFP-Cbl-c and CFP-Hic-5 were imaged directly after fixation. For immuno- staining focal adhesions, cells were permeabilized with PBS containing 0.1% saponin and 10% FBS for 5 min and incubated for 1 at room temperature or overnight at 4uC with the monoclonal anti-vinculin antibody (V9131; Sigma-Aldrich Corp., St. Louis, MO) at a 1:400 dilution. The antibody was diluted in PBS containing 0.1% saponin and 10% FBS and cells were washed using the same buffer twice for 10 min. The cells were then incubated with Donkey anti-mouse Cy3-labeled secondary antibody (165–150; Jackson ImmunoResearch Laboratories, Inc., Modulation of Cbl-c E3 Activity by LIM2 of Hic-5 West Grove, PA) for 1 h at room temperature. The slides were washed twice in PBS–0.05% saponin with 10% FBS and once in PBS. The cells were examined using a Zeiss LSM 510 META confocal microscope with a 636 /1.3 immersion-oil Plan- Neofluar objective (Carl Zeiss, Inc., Thornwood, NY). All the images were collected using the same pinhole, offset gain and amplifier values below saturation. The IMARIS software (version 6.1.5; Bitplane Scientific Software, South Windsor, CT) was used for colocalization analysis, and Pearson’s correlation coefficient (PCC) was calculated for each image analysis on 5–10 cells for each combination. In vitro E3 Assays In vitro E3 assays were performed in 30 ml reactions containing 50mM Tris-HCl pH7.5, 0.2 M ATP, 0.5 mM MgCl2, 0.1 mM DTT, and 1 mM phosphocreatine di(tris)salt (Sigma, St. Louis, MO), 15U creatine phophoskinase (EMD Biosciences, San Diego, CA), 50 ng purified recombinant rabbit ubiquitin-activating enzyme (662072; E1, EMD Biosciences, San Diego, CA), 0.5 mg ubiquitin (U100H; Boston Biochem, Cambridge, MA) and 1 ml of a crude bacterial lysate containing recombinant human UbcH5b (E2) [5,14]. GST-tagged Cbl proteins coupled to GSH-Sepharose beads or purified soluble Cbl-c were added to the reaction and assays were performed at 30uC, shaking at 1000 RPM. General incubation time was 1 h unless stated otherwise. Reactions were stopped by addition of SDS-PAGE protein loading buffer and boiling for 5 min. Where stated, 100 ng active Src (14–236; Millipore, Lake Placid, NY) was added to the reaction. Where stated, approximately 200nM Hic-5, Hic-5 C287A, or Hic-5 LIM2 was added to the reaction. Samples were resolved using SDS/PAGE and analyzed by immunoblots using anti-ubiquitin (Z0458, DAKO North America, Inc. Carpinteria, CA). Production and Purification of His Proteins Bacterial expression vectors were transformed in Rosetta bacteria (Invitrogen, Carlsbad, CA). Selected colonies were grown in LB broth with 100 mg/ml ampicillin selection. All His constructs were grown in 100 ml overnight cultures, diluted 1/ 10 in 900 ml of LB with 100 mg/ml ampicillin and grown at 37uC for 1 h. Protein production was induced with 2 mM isopropyl b-d- thiogalactoside at 37uC for 4 h. The culture was separated into 100 ml aliquots and pelleted by centrifugation. Extraction of His- tagged proteins from insoluble pellets was modified from a method previously described [40,41]. Cell pellets were resuspended in 10 ml of sonication buffer (20 mM Tris pH 8.0, 0.3 M NaCl, 1 mM EDTA, 1 mg/ml lysozyme) and incubated for 30 min on ice. Resuspended pellets were sonicated for 12 sec a total of 12 times and transferred to 1.5 ml microcentrifuge tubes and centrifuged at 10,0006g at 4uC for 10 min. Supernantants were pooled and saved. The His-tagged Cbl-c proteins were contained in the insoluble pellet. The individual pellets were washed three times in wash buffer (20 mM Tris pH 8.0, 1% Triton X-100, 5 mM EDTA) with centrifugation at 10,0006g at 4uC for 15 m between washes. Pellets were combined and solubilized in 1 ml lysis buffer (0.3% N-lauroyl sarcosine, 50 mM CAPS buffer pH 11.0, 0.3 M NaCl) at room temperature, rotated for 20 min and centrifuged 15 min at 10,000 g. The supernatant was removed and any remaining pellet was resuspended and treated as described above until no pellet remained after centrifugation. Combined supernatants were incubated with 1-ml slurry of Ni- NTA resin previously washed with lysis buffer (Qiagen, Valencia, CA) and rocked overnight at 4uC. The combined resin and sample were applied to a column, washed with lysis buffer plus 5 mM imidazole two times, 20 mM imidazole once and eluted with PLOS ONE | www.plosone.org 3 November 2012 | Volume 7 | Issue 11 | e49428