Los Ciclos Propedéuticos y las Competencias

8.3.1. Generation of GST-fusion proteins

Recombinant GST-cytoB1 was expressed in Escherichia coli TG-1. Fresh overnight LB- ampicillin (100mg/ml) cultures were diluted 10-fold with fresh medium and incubated at 37°C until the A600 was 0.4–0.5. Induction was for 6h at 23°C using 0.1mM isopropyl-β- D-thiogalactopyranoside (IPTG). The cells were harvested, washed with cold phosphate- buffered saline (PBS) and lysed in 50mM Tris, pH 7.5, 150mM NaCl, 1mM EDTA, 1mM phenylmethylsulfonyl fluoride (PMSF), 1mM benzamidine, 0.05% NP-40 and 0.5mg/ml lysozyme, for 30min at 4°C. After sonication and centrifugation at 10 000 g for 15min, the supernatant was mixed with glutathione–Sepharose (Pharmacia) for 30min at 4°C. Beads were washed in lysis buffer containing neither NP-40 nor lysozyme and the GST fusion proteins were eluted with 10mM glutathione in 50mM Tris, pH 7.5, 50mM NaCl and 1mM DTT.

Fractions containing the purified GST fusion proteins were dialyzed overnight against 20–50mM Tris, pH 7.5–8.0, 50mM NaCl, 0.1mM EDTA, 0.5mM DTT, 5% glycerol and stored in aliquots at –70°C.

8.3.2. Immunoblotting, immunoprecipitation and GST pull-down experiments

For immunoblotting, protein samples were separated by 10% SDS-PAGE or 15% Anderson PAGE and transferred to nitrocellulose membranes. For PTP-BL samples were separated by 6% SDS-PAGE. The membranes were blocked for 2h at room temperature in 7.5% non-fat milk in PBS plus 0.1% Tween-20. Anti-phosphotyrosine blots were performed using 4G10. For reprobing, membranes were stripped with 5mM sodium- phosphate buffer, pH 7.5, 2% SDS, 2mM ß-mercaptoethanol, where indicated.

For immunoprecipitation, cells were lysed in lysis buffer (50mM Tris-HCl, pH 7.5, 0.5- 1% Triton X-100, 150mM NaCl, 10mM NaPPi, 20mM NaF, 1mM sodium orthovanadate, 1mM PMSF, 2.5mM benzamidine and 10µg/ml each leupeptin and

aprotinin) and centrifuged at 10000g for 10min. For immunodepletion of SFKs, lysates from HUAECs were incubated with 2µg of anti-cst1 prebound to 20µl of proteinA- Sepharose. SFKs immunoprecipitations from cortical neurons were performed with monoclonal antibodies anti Src (2-17) and anti-Fyn (fyn-14) prebound to protein G- Sepharose 4B (Pharmacia). Polyclonal serum anti-ephrinB #23 was used for immunoprecipitation of ephrinB ligands from cortical neurons. Antibody #23 was covalently coupled to protein A-Sepharose as described (Harlow and Lane, 1988). Immunoprecipitations of ephrinB1 from NIH3T3-ephrinB1 cell lysates were done with a specific goat anti-ephrinB1 antibody. 4µg of antibody prebound to protein G beads were used per IP. After 2h at 4°C, immunoprecipitates were washed, denatured and analyzed by SDS-PAGE under non-reducing conditions.

For EphB2-Fc precipitation, cells were lysed in NP-40 lysis buffer (0.5 % NP40, 140mM NaCl, 1mM MgCl2, 1mM CaCl2, 1mM sodium orthovanadate, 20mM Tris/HCl pH 7.4 and protease inhibitors), diluted 1:1 with lysis buffer without NP-40. 2.5µg of EphB2-Fc, prebound to proteinG-beads, were used per precipitation. Precipitates were washed twice with diluted lysis buffer and bound proteins were analyzed for EphrinB and PTP-BL by SDS-PAGE and Western-blotting. For co-precipitation of PTP-BL with ephrinB1 10cm dishes of HeLa-B1 or HeLa-B1∆YKV cells were transfected with an expression construct encoding for a fusion protein consisting of the five PDZ domains of PTP-BL and an N- terminally fused EGFP. 48h later cells were lysed and subjected to EphB2-Fc precipitation as described above.

For GST pull-down experiments, 5µg of bacterially produced GST or GST-cytoB1 were prebound to 20µl of glutathione-sepharose beads and then incubated in E12.5 embryo head lysates (1 mg of protein). After 2h at 4ºC, the beads were washed three times in lysis buffer (50mM Hepes pH 7.5, 150mM NaCl, 10% glycerol, 1% Triton X-100, 1.5mM MgCl2, 1mM EGTA, 10mM NaPPi, 20mM NaF, 1mM sodium orthovanadate, 1mM PMSF, 2.5mM benzamidine and 10µg/ml each leupeptin and aprotinin) and analyzed by immunoblotting for the presence of PTP-BL.

8.3.3. Purification of membranes and rafts from mouse embryos and cortical neurons

For the analysis of detergent-insoluble complexes in flotation gradients, a membrane fraction was prepared at 4ºC as follows. Five heads from E12.5 mouse embryos or three 10cm dishes of cortical neurons were homogenized in a Dounce homogenizer with 1ml of ice-cold homogenization buffer (TNE: 25mM Tris pH 7.4, 150mM NaCl, 2.5mM EDTA containing 250mM sucrose, plus inhibitors as described above). The extract was passed five times through a 22G needle, ten times through 27G needle and centrifuged for 5min (3000 rpm, 4ºC). The supernatant was adjusted to 40% Optiprep and overlaid in a SW40 centrifuge tube with 7ml of 30% and 3ml of 5% Optiprep in TNE buffer. After centrifugation for 4h (24000 rpm, 4ºC; SW40 rotor), the floated membranes were collected in 600µl from the 5%/30% interface. Membranes were adjusted to 0.5% Triton X-100 for ephrinB1 and Src flotations and to 0.1% for PTP-BL flotations following extraction for 30min on ice. The extracted membranes were adjusted to 35% of Optiprep and overlaid in a SW60 centrifuge tube with 2.5ml 30% Optiprep and 400µl of 5% Optiprep in TNE buffer. After centrifugation for 4h (40000 rpm, 4ºC; SW60 rotor) seven fractions were collected from the top. After incubation with 1% Triton X-100 at room temperature, the fractions were immunoprecipitated with anti-ephrinB polyclonal antiserum (#23) for the detection of ephrinB ligands or total protein was precipitated with tricarboxylic acid (TCA) and analyzed by SDS-PAGE and Western-blotting for detection of Src and PTP-BL.

8.3.4. In vitro kinase assays

In vitro kinase assays with total lysates were performed in a final volume of 20µl of kinase buffer (80mM ß-glycerophosphate, pH 7.5, 20mM EGTA, 15mM MgCl2, 1mM DTT, 1mM PMSF, 2.5mM benzamidine and 2µg/ml each leupeptin and aprotinin) containing 4µg total protein from the lysate, 50µM unlabeled ATP, 10µCi of (γ-32P)ATP (5000 Ci/mmol) and 3µg of GST-cytoB1. After 30min at room temperature, the

phosphorylation reactions were terminated by addition of sample buffer and were analyzed by SDS-PAGE and autoradiography.

For kinase assays in pull-down experiments, 3µg of GST-cytoB1 or GST alone were prebound to 20µl of glutathione-Sepharose beads and then incubated in 1ml (1mg total protein) of cell lysates. After 2h rocking at 4oC, the beads were washed three times with kinase buffer and used for kinase assay in a final volume of 12µl of kinase buffer containing 50µM cold ATP and 10µCi of (γ-32P)ATP (5000Ci/mmol). After 30min at room temperature samples were analyzed by SDS-PAGE and autoradiography.

Kinase assays were also performed in immunoprecipitates from NIH3T3-ephrinB1 cells stimulated with Fc and EphB2-Fc. EphrinB1 was immunoprecipitated as described below and, after washing the immunocomplexes three times with kinase buffer, kinase assays were performed on the beads as described above for pull-down experiments.

8.3.5. Phosphoamino acid analysis

For phosphoamino acid analysis, GST-cytoB1 was phosphorylated in a pull-down assay from NIH3T3-ephrinB1 cells stimulated with EphB2-Fc. After SDS-PAGE, proteins were transferred to a PVDF membrane and the band corresponding to phosphorylated GST-cytoB1 was cut out. Phosphoamino acids were analyzed by HCl hydrolysis of the protein followed by thin-layer chromatography using standard procedures (Kamps, 1991).

8.3.6. In vitro phosphatase assay

GST-PTP-BL and GST-PTP-BL CysˆSer mutant were expressed in bacteria and purified as described (Erdmann et al., 2000). Tyrosine phosphorylated Src and ephrinB were immunoprecipitated from E12.5 mouse embryos with monoclonal antibodies anti Src (2-17) and polyclonal sera anti-ephrinB (#23) and the immunoprecipitates were incubated with 500ng GST-fusion proteins in 25mM Hepes, pH 7.5, 5mM EDTA, 10mM DTT. Immunoprecipitates were separated by SDS-PAGE and phosphotyrosine content

was determined using the anti-phosphotyrosine antibody 4G10 (UBI). For detection of phosphorylation of specific tyrosine residues in Src, anti-pY416 and anti-pY529 polyclonal antibodies were used. Total levels of Src phosphorylation and Src were detected using 4G10 and pan-Src polyclonal antibody respectively.

8.3.7. In gel kinase assay

For in gel kinase assays lysates were taken in 2x SDS-sample buffer supplemented with 10mM NaF, 0.4mM Na3VO4, 10mM EDTA pH8 and a cocktail of protease inhibitors. Samples were separated by SDS-PAGE on 10% Laemmli gels, which were polymerized in the presence of 100µg/ml of GST-cytoB1 or GST alone. After electrophoresis, the proteins were fixed by 2x 45min washing with 100ml 50mM Hepes pH7.6, 20% 2- Propanol. The gels were washed 2x30min in buffer A (50mM Hepes ph 7.6, 5mM β- mercaptoethanol). Proteins in the gel were denatured by incubation in 200ml of 6M urea in buffer A for 1h at RT. For renaturation gels were washed sequentially for 20min at 4°C in buffer A containing 0.05% Tween20 and 3M, 1.5M and 0.75M urea respectively, followed by 5 washes without urea at 4°C. Before kinase reaction gels were preincubated for 20min at 37°C under slight agitation in 30ml of kinase buffer: 40mM Hepes pH7.6, 20mM β-glycerophosphate, 20mM p-Nitrophenyl phosphate, 0.1mM Sodium Vanadate, 2mM DTT, 5mM MgCl2). Kinase reaction was performed under moderate agitation for 1- 1.5h at 30°C in 20ml of kinase buffer containing 50µM ATP and 100µCi (γ-32P)ATP. Kinase reaction was stopped by washing in 5% TCA, 1% Pyrophosphate, 2mM ATP buffered to pH 5.7 with Na2HPO4 (to prepare 5l approximately 263g are added). Gels were washed by changing the solution several times until radioactivity in the gel was barely around background.