Protein Methods

2.3.1 Western Blotting

Cells were harvested by trypsinization and centrifuged at 3,000rpm on a benchtop centrifuge for 2-5 minutes. The pellet was resuspended in 2x Protein Loading Dye (74 mM Tris-HCl pH 6.8, 1.25 mM EDTA, 20 % glycerol, 2.5 % SDS, 0.125 % bromophenol blue, 50 mM DTT) and mixed, before incubated at 95°C for 5-10 minutes. The sample was loaded on an SDS polyacrylamide (PAGE) gel, consisting of 13 % separating gel (4.33 ml 30 % acrylamide (37.5:1), 2.5 ml 4x resolving gel buffer (1.5 M Tris pH 8.8, 0.4% SDS), 33 µl 10% ammonium persulfate, 33 µl TEMED, water up to 10 ml) and 4 % stacking gel (1.7 ml 30 % acrylamide (37.5:1), 2.5 ml 4x stacking gel buffer (0.5 M Tris pH 6.8, 0.4 % SDS), 100 µl 10% ammonium persulfate, 10 µl TEMED, water up to 10 ml). Electrophoresis was carried out for 40 minutes at 200V in 1x Protein Running Buffer (25 mM Tris-HCl pH 8.3, 250 mM glycine, 0.1 % SDS (w/v)) in a BioRad Western Electrophoresis tank. The samples were transferred on a nitrocellulose membrane (Protran, GE Healthcare) for 1,5 hour at 65V in Western Transfer Buffer (25 mM Tris- HCl pH 8.3, 150 mM glycine, 10 % methanol) in a BioRad Western Transfer tank. The membranes were stained with Ponceau S solution (Sigma) confirming efficient transfer of the sample. Next, the membranes were blocked, in order to avoid any unspecific antibody binding, using 2 % non-fat milk (Marvel) in 1x Phosphate Buffer Saline

(PBS)/0.1 % Triton for 1-2 hours at room temperature. The membranes were then probed with the primary antibodies diluted in blocking solution (Table 2.8) overnight at 4°C before washed for 5-10 minutes for 3 times with 1x PBS/0.1 % Triton.

The membranes were subsequently probed with the secondary antibodies diluted in blocking solution (Table 2.9) for 1-2 hours at room temperature before washed again for 5-10 minutes for 3 times with 1x PBS/0.1 % Triton.

Antibody Source Reference # number Dilution

Donkey α-Goat HRP conjugated Donkey SantaCruz sc-2020 1 in 10,000

Donkey α-Mouse CD800 Donkey LICOR 926-32212 1 in 10,000

Donkey α-Mouse HRP conjugated Donkey SantaCruz sc-2314 1 in 10,000

Donkey α-Rabbit CD680 Donkey LICOR 926-60073 1 in 10,000

Donkey α-Rabbit HRP conjugated Donkey SantaCruz sc-2313 1 in 10,000

The membranes probed with Horseradish Peroxidase (HRP)-conjugated secondary antibodies were developed on an ECL film (GE Healthcare) using the Automatic Developer after the addition of ECL solution (Thermo Scientific). The membranes

Antibody Source Reference # number Dilution

α-CSL4 Rabbit

Watkins Lab, raised against

peptides, ref: 1390 1390 1 in 500

α-Dyskerin Rabbit SantaCruz sc-48794 1 in 1,000

α-FLAG Rabbit Sigma F7425-2MG 1 in 1,000

α-HA Mouse Perkeley Antibody Company N/A 1 in 5,000

α-Karyopherin Rabbit SantaCruz SC-11367 1 in 1,000

α-p21 Rabbit SantaCruz sc-397 1 in 200

α-p53 Mouse SantaCruz sc-126 1 in 500

α-PICT1 Goat SantaCruz sc-46615 1 in 500

α-PNO1 Rabbit

Watkins Lab, raised against

peptides N/A 1 in 500

α-RIO2 Goat SantaCruz E-14 E-14 1 in 200

α-RPL5 Rabbit SantaCruz A0912 1 in 5,000

α-RPL7 Rabbit Abcam ab72550 1 in 2,000

α-RPS19 Rabbit

P. Mason (Idol et al, 2007),

Sloan et al, 2013 (JCB) N/A 1 in 1,000

α-Tubulin Mouse Cell Signalling 3873 1 in 1,000

Table 2.8. The primary antibodies used for Western Blotting.

Table 2.9. The secondary antibodies used for Western Blotting.

probed with fluorescently-labelled secondary antibodies (CD680 or CD800) were visualized using the Odyssey-LICOR system (LICOR). Quantitation of the western blots was performed using the ImageQuant software (GE Healthcare), followed by normalization against the loading control.

2.3.2 Luciferase assay

U2OS cells expressing luciferase under the control of a p53-regulated promoter (kindly provided from Dr Neil Perkins, Newcastle University) were plated on a 24-well plate (approximately 5x104 _{cells) and treated appropriately before washed once with} Phopshate Buffer Saline (PBS) (Sigma Aldrich). The cells were harvested using 1x Lysis Buffer (Promega) and the luciferase reaction was carried out according to the manufacturer’s instructions using the Promega Luciferase Kit. Measurements of the luciferase intensity were taken using the Lumat 100 Luminometer (Berthold). Bradford assay was also performed in order to identify differences in cell numbers, where 10 µl of cell extract was added to 790 µl of water (800 µl of water was used as a blank control) before the addition of 200 µl of Bradford Reagent. The samples were incubated for 5 minutes at room temperature before the Optimal Density (OD) was measured at 595 nm using a Spectrophotometer (Ultrospec 2000). In order to calculate the fold difference in p53 activity, the luciferase intensity values were normalized against the OD values from the Bradford assay for each sample.

2.3.3 Glycerol gradients

10-40 % glycerol gradient (v/v) were prepared by adding approximately 2 ml of 10 % Glycerol solution (10 % Glycerol, 0.15 M KCl, 20 mM HEPES pH 8, 1.5 mM MgCl2, 1 mM DTT, 0.2 % Triton X-100) to an Ultra-Clear Centrifuge Tube (Beckman). Using a long needle, 40 % Glycerol solution (40 % Glycerol, 0.15 M KCl, 20 mM HEPES pH 8, 1.5 mM MgCl2, 1 mM DTT, 0.2 % Triton X-100) was under the 10% gradient solution. A rubber lid was placed at the top and the gradients were rotated at 83° angle for 1 minute and 10 seconds at 22rpm on a BioComp Gradient master (BioComp), before being stored in the fridge for 1h. In the meantime, approximately 5-7x106 _{cells were} resuspended in 0.5 ml of Gradient Buffer E (20 mM HEPES pH 8, 150 mM KCl, 0.5 mM EDTA, 0.1 mM DTT, 5 % Glycerol) on ice. The cells were disrupted by sonication twice for 15sec with a 30sec interval at 20 % amplitude/0.3sec pulse. 0.2 % Triton-X

100 was added to the samples before centrifuging for 10min at 4°C at 13,000rpm to remove any insoluble material. 400 µl were removed from the top of the gradient, which was then placed on a pre-cooled swTi60 bucket, where 400 µl of the extract were loaded. 40 µl of the sample before centrifugation were stored in -20⁰C, representing the 10 % total sample. The buckets were then balanced using Gradient Buffer E. Centrifugation took place in a swTi60 rotor (Beckman L7-80) for 1,5h at 52,000rpm at 4°C with brakes to slow acceleration. Each gradient was then fractionated using 200 µl from top to bottom and frozen in liquid nitrogen before being stored in -80°C. For Western Blot analysis, 20 µl of each fraction was added to 5 µl of 5x Protein Loading Dye and 5 µl of the total sample was added to 5 µl of 2x Protein Loading Dye, before loaded on an SDS-PAGE gel and analysed as described above.