Production of anti-PELO antibodies
Antibodies against the full-length of human PELO fused to GST (GST-PELO) were raised in rabbits (see below). Rabbits were initially injected with 1.0 mg of purified GST-PELO antigen mixed 1:1 with complete Freund's adjuvant followed by two boosts, at 3-weeks intervals, with 0.5 mg of antigen mixed 1:1 with incomplete Freund's adjuvant. The antibodies were affinity purified by absorption to the same fusion protein covalently coupled to HiTrap NHS-activated columns (Amersham Biosciences, Braunschweig, Germany) following the manufacturer's procedures.
HeLa cells were washed twice with PBS, and scraped in 0.5 ml of lysis buffer (50 mM Tris-HCl, pH 7.5, 2 mM EDTA, 2 mM EGTA, 1 mM sodium vanadate, 1 mM dithiothreitol, 0.1% Triton X-100, protease inhibitor cocktail) on ice for 15 min. The cells were homogenized with a glass Dounce homogenizer. The nuclear fraction was collected by centrifugation at 600 × g for 20 min at 4°C. The supernatant was further centrifuged at 100 000 × g for 60 min to collect a cytoplasmic soluble supernatant and a plasma membrane pellet. The nuclear and plasma membrane pellet was extracted with RIPA lysis buffer (Santa Cruz Biotechnologie, Heidelberg, Germany). To obtain a cytoskeletal fraction, cells were scraped on ice in cytoskeleton-stabilizing buffer (50 mM NaCl, 10 mM Pipes, pH 6.8, 3 mM MgCl2, 0.5% Triton X-100, protease inhibitor cocktail), and centrifuged at 14000 × g at 4°C. The pellet was resuspended in cytoskeleton-stabilizing buffer containing 0.5% SDS.
Yeast two-hybrid screening
To identify putative PELO-interacting proteins, human prostate cDNA library in pACT2 (BD Bioscience, Heidelberg, Germany) was screened using the pGBK7-PELO bait vector according to the manufacturer's instructions. Using sequential transformations, the bait vector and then the prey library were transformed into the AH109 strain. After the first transfection, the bait vector was characterized for lack of autoactivation by growing the PELO-transformants on plate lacking histidine and adenine. Double transformants were initially selected by growth on plates lacking histidine and adenine in the presence of 5 mM 3-amino-1,2,3-triazole (Sigma-Aldrich, Munich, Germany). From 1 × 107 transformants, 98 clones appeared after 5 days. These colonies were plated on plates lacking both histidine and adenine and containing β-galactosidase. Approximately 56 of those displayed clear activation of all three reporter genes, and 42 of these were selected for isolation of the prey plasmids and sequencing of the cDNA encoding the PELO-interacting protein.
Expression of proteins in mammalian cells
For transient expression, human HeLa cells were grown for 24 h on 6-well plates (1.5 × 105 cells/well) prior to transfection with recombinant construct (2 μg/well) using Lipofectamin 2000 transfection reagent (Invitrogen, Karlsruhe, Germany) according to the manufacturer's procedures. Transfected cells were further cultured for 2 days prior to harvesting for co-immunoprecipitation studies. For stable expression, HepG2 cells were transfected either with pcDNA™ 3.1/Myc-His A(+) vector alone (Invitrogen) or with recombinant vector containing the full-length PELO cDNA, which was in-frame with the Myc tag. The cells were trypsinized 48 h after transfection, and cultured in medium containing 600 μg/ml of G418. After 2 weeks, single colonies were picked, cultured and the expression of Myc-tagged PELO was analysed by immunoblotting using Myc tag-specific monoclonal antibody (1:200 dilution, Santa Cruz Biotechnologie).
Immunoprecipitation and Immunoblotting
The plasmid constructs Myc-HAX1, Myc-EIF3G and Myc-SRPX, which were used in co-immunoprecipitation experiments, were generated by amplification the cDNA fragments of HAX1, EIF3G und SRPX using the identified prey vectors as template and the sense primer Y2HF: 5'-TTC GAT GAT GAA GAT ACC CCA AAC-3' and antisense primer Y2HR: 5'-GTG AAC TTG CGG GGT TTT TCA GTA TCT AC-3'. The amplified PCR fragments were inserted into the Eco RI/Xho I sites of the pCMV-Myc vector (BD Bioscience). The PELO-HA construct was generated by insertion of cDNA fragment of full-length human PELO into the Eco RI/Xho I sites of the pCMV-HA vector (BD Bioscience), which had a HA epitope linked to the N-terminus of PELO protein. HeLa cells were transiently co-transfected with PELO-HA and either of the Myc-HAX1, Myc-EIF3G or Myc-SRPX constructs. For co-immunopreciptation, 500 μl of whole cell lysates were firstly pre-cleared by incubation with 50 μl protein A/G-agarose beads (Roche, Mannheim, Germany) for 3 h. The pre-cleared lysates were then incubated with either 10 μl mouse monoclonal anti-Myc (Santa Cruz Biotechnology), 10 μl rabbit polyclonal anti-HA (BD Biosciences) or 4 μl polyclonal anti-PELO antibodies for 2 h at 4°C. In control assays, lysates were subjected to immunoprecipition using 4 μl of mouse IgG. The immunoprecipitates were recovered by incubating the mixture overnight at 4°C with 50 μl protein A/G-agarose beads. After extensive washing of the beads, bound proteins were separated by SDS-PAGE and transferred to nitrocellulose membrane Hybond-C (Amersham Biosciences). Membranes were blocked for 1 h in 5% non-fat dry milk in PBS containing 0.2% Tween20 and then incubated overnight at 4°C with either anti-Myc, anti-HA or anti-PELO antibodies (1:500 dilution). Bound antibodies were then visualized by secondary incubation with goat anti-rabbit IgG horseradish peroxidase conjugate or goat anti-mouse IgG horseradish peroxidase conjugate (Sigma-Aldrich) followed by SuperSignal® West Pico Chemiluminescent Substrate (Pierce, USA).
GST pull-down assay
For generation of glutathione S-transferase-PELO and truncated fusion constructs, cDNA fragments encoding full-length human Pelo (GST-1, 2-385 aa), and truncated Pelo - GST-2 (136-385 aa), GST-3 (2-371 aa) and GST-4 (2-268 aa) were amplified by PCR using human Pelo cDNA as template and appropriate primers containing restriction site tags. Oligonucleotide sequences are given in Additional file 1. The amplified fragments were cloned in-frame into Eco RI/Xho I sites of the pET41a(+) vector (Novagen, Darmstadt, Germany). B121(DE3)pLysS bacteria were transformed with either empty GST vector (GST-0) or GST-PELO fusion vectors and grown at 37°C to log phase (A600 of 0.6). Expression of fusion protein was induced by growing the cells at 30°C for 4 h in the presence of 100 μM 1-thio-β-D-galactopyranoside. GST-PELO fusion proteins were purified using the GST-bind™ Purification Kit (Novagen, Darmstadt, Germany). HeLa cell protein extracts (500 mg protein) were incubated with 50 μl (100 μg) of individual fusion protein or GST alone in the GST binding buffer for 3 h at 4°C. After centrifugation, pellets were washed 3 times with lysis buffer B, boiled in sample buffer, followed by SDS-PAGE and Western blot analysis.
100 μl of GST-bind-resin were transferred to a clean 1.5 ml microcentrifuge tube and washed 3 times with 250 μl of 1× GST Bind/Wash buffer. The beads were resuspended in 50 μl 1× GST Bind/Wash buffer and incubated with 50 μl (100 μg) of individual GST fusion protein or GST alone for 1 h at RT on a rocking platform. Beads were washed 3 times with 250 μl of 1× GST Bind/Wash buffer and 100 μl of HeLa cell lysates (300 - 500 μg protein) were added to the reaction tube and the mixture was incubated for 2 h at 4°C on a rocking platform. The beads were then washed 3 times with 250 μl of lysis buffer B. Finally, proteins binding to the beads were resuspended with NuPAGE SDS sample buffer, separated by SDS-PAGE and analyzed by Western blotting using anti-c-Myc primary antibodies (dilution 1:200) and goat anti-mouse IgG conjugated with horseradish peroxidase secondary antibodies. Immunoreactive polypeptides were then visualized by the SuperSignal® West Pico Chemiluminescent Substrate as previously described.
Spreading, proliferation and soft agar assays
HepG2 cells stably transfected with empty pcDNA3A-Myc or pcDNA-PELO plasmids were seeded at a density of 2 × 104 cell/well onto 96-well plates that were pre-coated with 10 μg/ml fibronectin (Sigma-Aldrich). Cells were allowed to spread for 60 min at 37°C in growth medium. Non-adherent cells were removed by two washes with pre-warmed PBS. The percentage of spreaded cells was evaluated 30 min and 120 min after seeding by counting the number of non-spread cells under the microscope (Olympus BX60, Hamburg, Germany). The non-spread cells were defined as round cells, whereas spread cells were defined as those that lacked a rounded shape and had extended membrane protrusions . To obtain an index of cell spreading, adherent cells were photographed with a digital camera. According to their distinct differences in surface area and morphology, the cells were divided into two groups: small round cells showing no obvious sign of spreading (group 1), and flat cells in the process of spreading or already well spread (group 2). Monitoring the proportion of cells in group 1 to total cells at a given time point after seeding serves as a measure of spreading velocity. At least 100 cells per well were counted in three independent experiments.
For cell proliferation assay, stably transfected HepG2 cells with empty pcDNA3A-Myc or pcDNA-PELO plasmids were seeded in triplicate in 24-well plates (0.25 × 105 cells per well) and cultivated for 5 days. Every day, cells were washed, trypsinized and proliferation of cells was evaluated by counting the number of cells under the microscope using a Neubauer counting chamber.
For soft agar colony formation assay, single cells of stably transfected HepG2 cell were resuspended in semi-solid medium containing 0.35% Bacto-agarose supplemented with 20% FBS, 750 μg/ml G418 and 0.8% agarose. This cell suspension containing 0.2 × 104 cells/well was immediately plated into 6-well plates coated with 0.3% agar in cell culture medium (2 ml per well) and cultured at 37°C with 5% CO2. After 3 weeks, the top layer of the culture was stained with 0.1% crystal violet for 1 h. The culture was analyzed in triplicate and colonies larger than 100 μm in diameter were counted.
Cells were grown on chamber slide overnight, fixed with 4% paraformaldehyde in PBS for 15 min. Subsequently, cells were washed with PBS, preincubated with 0.2% Triton X-100 in PBS for 30 min and blocked with 10% goat serum in PBS for 1 h. Cells were incubated overnight at 4°C with 100 μl of primary antibody diluted in 1% goat serum in PBS. Primary antibodies were as followed: a 1:200 dilution of anti-PELO antibody, a 1:200 of anti-Myc and anti-HA antibodies. Cells were washed with PBS and then incubated with a 1:500 dilution of the appropriate FITC-coupled secondary antibody (Sigma-Aldrich) in 1% goat serum in PBS for 2 h. Cells were washed and then incubated with 1:100 dilution of FITC-phalloidin (Sigma-Aldrich) in PBS for 20 min. Cells were mounted with Vectashield mounting reagent (Vector, Burlingame, USA) prior to fluorescence microscopy (Olympus, Hamburg, Germany).
Bimolecular fluorescence complementation (BiFC) assay
The pEGFP-N1 (BD Bioscience) and pQM-Ntag/B (Abcam, UK) vectors were used as start plasmids to construct the BiFC vector FPCA-V1 and FPCA-V2, respectively. To generate the FPCA-V2 and FPCA-V1, fragments containing the coding sequence for the residues 1-157 or the residues 158-239 of the EGFP fragment were amplified by PCR and inserted into the backbone of the pQM-Ntag/B and pEGFP-N1 vectors, respectively. The full-length cDNA fragment of human Pelo (1-385 aa) was amplified and cloned into the Xho I/Bam HI-digested FPCA-V1 to generate PELO-GFPC construct. The cloned cDNAs of HAX1 (178-279 aa), EIF3G (1-320 aa) and SRPX (265-464 aa) were amplified by PCR and inserted into Hind III/Bgl II-digested FPCA-V2 to generate HAX1-GFPN, EIF3G-GFPN and SRPX-GFPN constructs, respectively. Primer sequences used for generation a cDNA fragments are presented in Additional file 1. HeLa cells grown on chamber slides were transiently co-transfected with PELO-GFPC and either HAX1-GFPN, EIF3G-GFPN or SRPX-GFPN constructs. Transfected cells were grown at 37°C for 24 h, incubated for 2 h at 30°C and then fixed in 4% paraformaldehyde in PBS for 10 min. Single transfection of the plasmids used for BiFC (PELO-GFPC and HAX1-GFPN) did not produce any fluorescence.