Investigaciones empíricas

2.3.1 Cell culture

HeLa cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 µg/ml penicillin, 100 µg/ml streptomycin and 2 mg/ml L-glutamine. SKBR3 cells were grown in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% FBS, 100 µg/ml penicillin, 100 µg/ml streptomycin and 2 mg/ml L-glutamine. HEK293/ALK stable cells were grown in DMEM supplemented with 10% FBS, 100 µg/ml penicillin, 100 µg/ml streptomycin, 2 µg/ml puromycin and 2 mg/ml L-glutamine. Cells were incubated at 37 °C in a humidified atmosphere containing 5% carbon dioxide.

2.3.2 GST-NUMB-PTB expression, purification and biotinylation

The BL21 strain of Escherichia coli was transformed with pGEX6P3-dNUMB-PTB or pGEX6P3-mNUMB-PTBL/PRBS. Positive colonies were cultured in Lysogeny Broth (LB) medium to a density of OD600 0.6~0.8, then the protein expression was induced with 0.5 mM IPTG for 16 hours at 18°C. The bacterial cells were harvested and pellets were re- suspended in PBS buffer containing complete protease inhibitors (Roche). Triton X-100 was added to a final concentration of 2%, lysozyme was added to a final concentration 1 mg/ml and benzonase was added to a final concentration 20 units/ml. The suspension was sonicated six times (10 seconds each) on ice and then lysed for 30 min at room temperature. Lysates were centrifuged at 15,000 × g for 30 min at 4°C and the supernatant was collected. Purification of GST-tagged proteins was performed with glutathione resin (GE healthcare). The resin was washed with PBS buffer three times. The lysate supernatant was loaded to the resin followed by three column volume washes with PBS buffer, then eluted with 10 mM glutathione in PBS buffer (no elution step for pulldown assay). To determine GST- protein purity, a small amount of purified protein (or protein on resin) was boiled with SDS-loading dye and then analyzed by SDS-PAGE/Coomassie staining. The GST NUMB- PTB domains used for the Protoarray hybridization were biotin-labeled using EZ-Link™ Sulfo-NHS-Biotinylation Kit (Thermo) in 1×PBS buffer.

2.3.3 ProtoArray hybridization

The Protoarray Human microarray (Invitrogen) was equilibrated at room temperature for 30 minutes prior to blocking. The array was arranged barcode-side up in each well of a chilled 4-chamber incubation tray. 5 ml blocking buffer (50 mM HEPES pH 7.5, 200 mM NaCl, 0.08% Triton X-100, 25% Glycerol, 20 mM Reduced glutathione, 1 mM DTT and 1× Synthetic Block) was then added to the array and incubated for 1 hour at 4 °C with slow shaking. After blocking buffer was removed, biotinylated GST-dNUMB-PTB was diluted to final concentration of 5 µM in washing buffer (1×PBS, pH 7.4, 1×Synthetic Block, 0.1% Tween 20) to form the blotting solution. 120 µL of blotting solution was added on top of the array surface and then a LifterSlip cover glass was carefully attached to the array. The array was blotted for 90 minutes at 4 °C without shaking. The cover glass was then removed and the array was washed with washing buffer 5 times for 5 minutes each at 4 °C with slow shaking. The array was visualized by incubation with 5 ml Alexa Fluor 647- conjugated streptavidin for 90 minutes at 4 °C with gentle shaking. Then the array was washed again following the same procedures as above. Finally, the array was dipped into distilled water once to remove salt residues and scanned using Tecan fluorescence microarray scanner. Data were analyzed using ArrayPro software.

2.3.4 Transient transfection

Plasmid DNA was first diluted in serum-free Minimum Essential Media (MEM) to a final concentration of 0.01 µg/µl. For each 10-cm dish, 40µl X-tremeGENE HP DNA transfection reagent (Roche) was mixed with 1ml plasmid DNA in MEM. The mixture was added to the culture dish (10ml) after 15 min incubation at room temperature. After transfection, cells were incubated for 48~72 hours to allow for ectopic gene expression.

2.3.5 Immunoprecipitation, pull down and western blotting

Cultured cells were lysed in ice-cold mammalian cell lysis buffer (1% Triton X-100, 50 mM Tris-pH 7.2, 150 mM NaCl, 2 mM MgCl2, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM DTT, 10 mM NaF) containing complete protease inhibitors (Roche). Cell debris was removed by centrifugation, and 500 µg of supernatant protein was incubated for four hours

in the presence of 1 µg antibody and 30 µl of 50% slurry protein G-Sepharose beads (Roche), or 1 µg immobilized GST fusion protein (beads). The beads were subsequently washed three times with lysis buffer and boiled with SDS-loading dye. The precipitates were resolved by SDS-PAGE. Proteins were then transferred to polyvinylidene fluoride PVDF membrane using semi-dry transferring method, and detected by immunoblotting with appropriate antibodies and visualized by enhanced chemi-luminescence (ECL).

2.3.6 Free peptide and peptide array synthesis

Both free and membrane-bound peptides were synthesized using an automatic Intavis AG workstation from amino acid monomers protected with Fmoc (9-fluorenylmethyl- oxycarbonyl). Rink-resin (Rink-NH2) was used to couple the first amino acid in free peptide synthesis, whereas the amine-derivate cellulose membrane (cellulose-NH2) was made to couple the first amino acid in on-membrane peptide synthesis. In each synthesis cycle, the carboxyl group of Fmoc-protected amino acid (Fmoc-R-COOH) was first linked to the amine group of the previous amino acid (or Rink-NH2) through an amide bond. All unoccupied amine groups were then blocked (acetylated) by acetic anhydride to prevent incorrect amide bonds forming in subsequent cycles. Next, the Fmoc group was removed by piperidine (de-protecting) to release the free amine group primed for linking the carboxyl group of next amino acid residue. Fluorescein-NHS was linked to the amine group of the last amino acid in free peptide synthesis. After synthesis, the on-membrane peptides were treated with a mixture containing 47.5% TFA (trifluoroacetic acid), 1.5% TIPS (tri- isopropylsilane) and 51% water, to remove all other protecting groups on amino acid side chains. The free peptides were treated with a mixture containing 95% TFA, 3% TIPS and 2% water to de-protect side chains while cleaving the peptide from Rink-resin.

2.3.7 Far western assay for peptide membrane blotting

The peptide membrane was blocked with 5% skim milk in TBST buffer (0.1 M Tris-HCl, pH 7.4, 150 mM NaCl, and 0.1% Tween 20) for 1 hour at room temperature, with slow shaking. GST-fusion proteins were added directly into the blocking buffer to reach a final concentration of 1µg/ml and incubated with the peptide array membrane for 1 hour at room

temperature, with slow shaking. The membrane was then washed with TBST buffer three times for 5 minutes each with shaking. Next, anti-GST-HRP antibody was added at 1:3000 (v/v) into 5% milk in TBST buffer. The membrane was incubated for 1 hour at room temperature with slow shaking followed by the same washing procedures as above. Finally, the bound GST-protein was visualized using ECL.

2.3.8 Determination of dissociation constant

The dissociation constant between protein and peptide was determined using a fluorescence polarization (FP) assay. Proteins and fluorescein labelled peptides were prepared following the procedures introduced above. To reduce variation, two independent tests were prepared simultaneously. 384-well flat bottom plates (Corning-3537) were used for the assay and a PerkinElmer Envision 2103 plate reader was used for evaluating FP. The peptides (approximate 2 µM each, mass) were dissolved in 100 µl DMSO and 3µl was diluted 100 fold in water. The concentrations of the proteins were determined and adjusted to 50 µM. 2-fold serial dilutions were set up to result in 16 different protein concentrations ranging from 0 to 50 µM. 30 µl of each protein sample was mixed with 5 µl peptide in the 384-well plate and the FP was measured. Non-linear fit was approximated by assuming peptide concentration was much lower than Kd value in the following equation:

∆FP = FPobs - FP0 = FPmax x [domain] / (KD + [domain])

The full equation without the approximation can be found in this study (Kaushansky et al., 2010).

2.3.9 Consensus binding motif and SMALI matrix

The OPAL membrane was scanned and the intensity was quantified on a Bio-Rad imaging system. To generate consensus motifs based on specific binding signals, the average background signal of the membrane was subtracted from the dataset. Since a conspicuous column of Lys or Arg was detected in the corresponding OPAL screen, these strong Lys or Arg signals are position-independent. To minimize position-independent effect, the

value for each Lys or Arg spot was readjusted by subtracting the average of the Lys or Arg in the column. See more details for SMALI scoring in (Li et al., 2008b).