Internalisation assay and flow cytometry analysis

Transfected Jurkat cells (refer to section 2.6.1; page 55) (untransfected cells as negative control) were re-suspended in 1 mL of 0.5% BSA/PBS to give cell density of 1 X 10⁶ per mL. Cells were washed with 0.5% BSA/PBS twice and re-suspended with 300 µL of 0.5% BSA/PBS. Cells were activated with 15 nM CXCL12 at 37^oC for 15 minutes or left untreated as negative control. Cells were then washed with ice-cold 0.5% BSA/PBS twice and were re-suspended in anti-h CXCR4 antibody (R&D systems) (1 in 2000) at 4^oC for 1 hour (resuspension with 0.5% BSA/PBS as negative control). Cells were gently washed twice in ice-cold 0.5% BSA/PBS and then incubated at 4^oC for 1 hour with secondary antibody goat anti-Mouse IgG (H+L), F(ab’)2 fragment, CF 647 (Sigma Aldrich) (1 in 200). Stained cells were gently washed twice with 0.5% BSA/PBS and gated to exclude dead cells. Differences in cellular fluorescence were then determined and quantified using BD FACSCalibur (BD Biosciences) and data analysed using CellQuest software version 3.1 (Becton Dickinson, San Jose, CA).

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2.12. Western blotting

2.12.1. Sample preparation

5 X 10⁵ cells were spun down at 1800g for 5 minutes and the supernatant was removed.

Cells were then either frozen as a pellet for later analysis or used immediately. On the day of analysis, the pellet was defrosted (where necessary) and 40 µL of lysis/ sample buffer (Mammalian Protein Extraction Buffer, GE Healthcare Life Sciences) was added and mixed until a homogenous consistency was reached. Mixture was incubated in ice for at least 30 minutes to promote cell lysis. The lysis mixture was then immediately spun down at 14000g for 10 minutes. The supernatant was removed in preparation for gel electrophoresis and the pellet was discarded. Protein concentration of samples were then measured by the Nanodrop spectrometer system (ThermoFisher Scientific). The lysed sample was placed in boiling water for 5 minutes to denature proteins further before use for SDS-PAGE.

2.12.2. SDS-PAGE sample separation

SDS-PAGE gels consisted of a stacking phase for sample loading (0.1089g Tris, SDS 2%

pH 8.8, 4.5% acrylamide v/v, 0.1% ammonium persulphate w/v, 0.01% TEMED v/v) which constituted the top 20% gel and a 12% acrylamide running phase (0.67155g Tris/SDS 2% pH 8.8, 12% acrylamide v/v, 0.1% ammonium persulphate w/v, 0.01%

TEMED v/v). 20 µL of sample was loaded into the stacking phase wells with a Hamilton microsyringe along with 5µL Pierce pre-stained protein MW marker (ThermoFisher Scientific) as ladder. Unused wells were loaded with 20µL loading buffer (4% SDS, 0.02% bromophenol blue, 20% glycerol, 80 mM Tris, pH 6.8) to allow identification of the elution front. Gels were run at a constant 30 mA until the elution front reached the running gel when the current is increased to 50 mA constant until the elution front ran off the gel, at which point electrophoresis was complete.

2.12.3. Protein transfer

The gel plates were removed from the clamp assembly and separated so that the gel remained intact on one plate. The gel was then transferred to a nitrocellulose

64 membrane (Whatman) pre-soaked with transfer buffer. The membrane and gel were placed between sheets of (Whatman) filter paper pre-soaked in transfer buffer (25 mM Tris, 192 mM glycine, 10% methanol, pH 8.3), sufficient sheets were used to allow a good contact between electrodes of the transfer plate in the semi-dry blotter kit (Sigma-Aldrich). The transfer was undertaken at 15 volts constant for 45 minutes.

Successful protein transfer was evaluated by the transfer of the protein marker to the nitrocellulose membrane.

2.12.4. Immunostaining and imaging

After protein transfer was verified the nitrocellulose membranes were initially blocked for non-specific binding with PBS-T (0.5% v/v Tween-20 in PB) containing 5% w/v low-fat milk powder (Marvel) for 1 hour at room temperature. Blocking buffer was then removed and the relevant primary antibody (table 2.4) was diluted in blocking buffer before overnight rolled-agitation at 4°C. The following day the primary antibody was removed, and the membrane washed 3 times for 5 minutes with PBS-T before addition of the relevant secondary peroxidase tagged Ab was added at 1:5000 in 10 mL blocking buffer. After 1-hour roller-agitation, the secondary antibody solution was removed, and the membrane was washed 3 times for 5 minutes in a tray with gentle shaking to facilitate flattening. Proteins were visualised by addition of Pierce ECL Western Blotting Substrate (ThermoFisher Scientific) after which the membrane was sandwiched between 2 acetate sheets and imaged using the G-Box imaging system (Syngene, UK) set to automatically calculated exposure. Images were captured using Gene-snap software (Syngene, UK).

2.12.5. Membrane stripping

Antibodies were removed from the membrane before a second stain by incubation with Millipore Stripping Solution (Millipore, Temecula California) at room temperature for 15 minutes before blocking and re-probing for immunostaining as described previously (section 2.11.4).

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2.13. Bioluminescence Resonance Energy Transfer (BRET)

Cells were transfected with plasmids DNA CXCR4-RLuc3 (donor) and h-arrestin3-GFP10 (acceptor) and also maintained as described previously (refer to section 2.6.1;

page 55). Transfected cells were then seeded in 96-well plates (with clear bottoms that had been treated with poly-D-lysine) (Corning) and left in culture for 24 hours before being processed for BRET assay. Transfection was assessed by visualising localisation of fluorescent h-arrestin3-GFP10 in cells using Leica DMII fluorescence microscope with GFP filter cube. Luciferase substrate solution, coelenterazine 400a (DeepBlueC) (Biotium), stock was reconstituted in ethanol for BRETassay and EnduRen (Promega) stock was reconstituted in DMSO. For routine BRET measurements, cells were washed with BRET buffer (PBS, 0.5 mM MgCl2, 0.01% BSA) 24 to 48 hours after transfection and luciferase substrate solution, DeepBlueC (Biotium) or EnduRen (Promega) was added to a final concentration of 5 µM or 60 µM respectively in BRET buffer (PBS, 0.5 mM MgCl2, 0.1% glucose). DeepBlueC (Biotium) was added just before detecting emission from that well and EnduRen (Promega) was added to live cells at least 1.5 hours before BRET detection. Readings were then collected using Clariostar Monochromator (BMG Labtech) measuring signals detected in the 410±40 nm and 515±15 nm windows for luciferase and GFP10 light emissions. The BRET signal is determined by calculating the ratio of the light intensity emitted by the h-arrestin3-GFP10 over the light intensity emitted by CXCR4-RLuc3. The values were corrected by subtracting the background BRET signal detected when the CXCR4-RLuc3 construct was expressed alone. To assess the effects of ligands, CXCL12 was added at the 0.01 nM, 0.1 nM, 1 nM, 5 nM and 10 nM and incubated at 37^oC for 5 minutes before the addition of luciferase substrate solutions and BRET reading. For acquisition of full BRET spectra, cells were transfected as described above with different amounts of h-arrestin3-GFP10 for a given quantity of CXCR4-RLuc3 (0.1µg). Cells were detached and re-suspended in BRET buffer (PBS, 0.5 mM MgCl2, 0.01% BSA). 200,000 cells were seeded in 100 µL of BRET buffer (PBS, 0.5 mM MgCl2, 0.01% BSA) in a 96-well plate with a clear bottom (Corning), and BRET scan was performed in FLUOstar Optima (BMG Labtech) by reading luminescence between 480 – 520 nm after addition of DeepBlueC for cells expressing [acceptor]/[donor] ratios.

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2.14. Curve prediction and statistical analysis

All data were analysed using GraphPad Prism 6 and represent at least 3 independent experiments (unless stated otherwise). All concentration response sigmoidal curves used to calculate EC50 and IC50 values were fitted assuming a Hill coefficient of 1.

Statistical significance was determined using unpaired student t-tests where applicable or one-way ANOVA analysis of variance followed by Bonferroni or Dunnett’s multiple comparison as post-tests where 3 or more data sets were to be analysed. A significant value of 95% was used with p values indicated as follows p≤0.05 = *, p≤0.01 = **, p≤0.001 = ***. P values above 0.05 were therefore considered as not significant (ns).

All error bars represent standard error of the mean value (S.E.M).

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