Immunofluorescence on fixed cells

2.14.1 Cell line maintenance

All the cell line culture was performed in a NuAire Labgard 437 ES Class II Biosafety Cabinet using the standard aseptic technique. When required, cells were removed from a -80°C freezer, thawed by immediate swirling in a 37°C water bath, and then rapidly transferred into a tube containing 19ml of Dulbecco's Modified Eagle Medium (DMEM), from Lonza, supplemented with 10% Fetal Bovine Serum (Sigma-Aldrich). The cells were then collected by centrifugation at 3000rpm for 3 minutes, decanting the media, and resuspension in 1 ml DMEM. Cells were grown in T75 flasks (Corning) containing DMEM in a Sanyo MCO 20AIC cell culture incubator set at 37°C and 5% CO2. Cells were passaged after their confluency by dissociating them from the surface of the flask with 10 ml of 0.25% trypsin (Lonza) and 2-10 minutes incubation at 37°C and 5% CO2. Sufficient banging into the flask for about 1-2 minutes ensured the maximum removal of the cells attached to the inner flask wall. Using a pipette, the flask wall was flushed thoroughly using the same suspension of cells in the flask and all the cells were transferred into a new Falcon tube containing 15ml of DMEM. The tube containing the 25 ml cell suspension was inverted 3 times to achieve proper mixing of cells. Subsequently, 3 minutes centrifugation at 3000rpm was given to the tube and the media was discarded, leaving the cell pellet, which was resuspended in 1ml of DMEM. Then, the cells were vigorously mixed by pipetting up and down about 20 times before adding 9 ml of fresh DMEM. So, the cells at this stage were suspended in a total of 10ml of DMEM.

2.14.2 Cell counting

For accuracy and consistency, the resuspended cells were counted before their seeding into new flasks. The count was carried out by mixing 50μl of trypan blue stain (Thermo Fisher Scientific) with 50μl of the cell suspension, and then loading into a clean haemocytometer. The haemocytometer was placed under an Olympus CKX41 bright field microscope and cells were counted. The resuspended cells were then diluted based on the desired seeding density.

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2.14.3 Transfection

Transient DNA transfection was done to assess the over-expression of plasmids in cultured SHSY5Y cells. Plasmid transfection was carried out using an Effectene Transfection Kit (Qiagen), according to the manufacturer’s instruction. The cells were first plated overnight in 24-well plates containing sterile coverslips (Fisher Scientific) in each of the wells. The next day, transfection was done when the growth was about 60-80% confluency. The cells were transfected while growing at 37°C with 5% CO2 for 16 hours at 0.2μg concentration using 5 μl of Effectene Transfection Reagent and 1.6μl of Enhancer, all diluted in 400μl of DMEM.

After 16 hours of incubation, the media (with the transfection complex) was removed and the cells were immediately resuspended in 500μl of fresh warm (37 oC) DMEM media to prevent cell toxicity.

2.14.4 Cell fixation

The cells were washed three times with 500μl of warm (37 o_{C) Dulbecco's} phosphate-buffered saline (DPBS, from Sigma) on a rocking platform (Roto- Shake Genie, Qorpak, USA) for 1 minutes at RT. After that, they were fixed with 500μl of 4% paraformaldehyde (Alfa Aesar, UK) for 10 minutes on the Roto- Shake machine. The paraformaldehyde was immediately removed, and 1ml of DPBS containing 50mM NH4Cl (1.5g in 500ml) was added to the cells, which were then rocked for 1 minute to quench excessive fixative and prevent any subsequent non-specific cross-linking later. The cells were then washed three times with 500μl of DPBS (Lonza) and 5 minutes of rocking.

2.14.5 Cell permeabilisation

Cell permeabilisation was carried out using 0.2% triton X-100 (Sigma-Aldrich) with rocking for 4 minutes, then rinsing the cells twice with 500μl DPBS. The cells were then blocked in 500μl DPBS containing 3% donkey serum (Sigma-Aldrich) for 2 hours with rocking at RT. After that, the cellular coated coverslips were carefully transferred into a new 24-wells plate.

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2.14.6 Immunostaining

To each well, 250μl of the appropriate primary antibody, diluted in blocking solution containing 3% donkey serum, was added. Goat anti-whirlin antibody (Santa Cruz, sc-49787) was used at a 1:100 dilution and rabbit anti-UBR4 antibody (Abcam, ab86738) was used at a dilution of 1:250. The plate was transferred into a moist chamber and incubated overnight at 4°C on a rocking platform. The next day, the cells were washed three times with 500μl of 0.1% tween solution (Bio-Rad) for 5 minutes on a rocking platform at RT. The cells were then incubated for 1 hour with 250μl of fluorescently conjugated secondary antibodies in blocking buffer with rocking at RT. Both secondary antibodies (Anti- Goat 488 and Anti-Rabbit 488; Invitrogen) were used at a dilution of 1:500 dilution. The cells were then washed in 500μl DPBS for 10 minutes with rocking before their nuclei were stained with 500μl of 1:1000 diluted DAPI (Sigma- Aldrich). The cells were then given two final washes with 500μl DPBS for 10 minutes with rocking at RT.

2.14.7 Slide mounting

The correct number of microscopic slides was labelled and about 50μl of Fluoromount-G® mounting medium (Southern Biotechnology) were added at the centre of each slide. Then, the correct coverslips were carefully taken from the 24-well plate and mounted upside down on the mounting medium. Any air pebbles present between the slide and the coverslip were driven out by gently pressing on the coverslip. The slides were then kept for at least 12 hours at 4°C before they were taken for confocal microscopy.

2.14.8 Confocal microscopy

The slides were analysed and imaged using both Zeiss LSM510 and Nickon Eclipse TE2000-E microscopes. Each of them has its own associated imaging software. In general, confocal imaging involved using 408nM, 488nM and 543nM lasers for exciting DAPI, Alexa Fluor® 488 and Alexa Fluor® 563 fluorophores, respectively. Multiple photographs were taken for each view of interest using different cellular slices, and co-localization was assessed on 30 cells using the

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ImageJ software open source image processing package (http://fiji.sc/). The confocal microscopy and Fiji ImageJ assessment was also independently done by Dr James Dachtler, faculty of biological sciences, university of Leeds.

2.15 Co-immunoprecipitation

2.15.1 Brain tissues homogenising

For minimal disturbance of hypothesised interaction between whirlin and UBR4, ultrasonication was selected as the method for homogenising right brain hemispheres collected from three different wild type adult mice. The tissues were first weighed and then Protease Inhibitor Cocktail (cOmpleteTM_{, EDTA-free;} Roche) was added to each tissue based on its weight, 5ml of inhibitor per 1g of tissue. Each sample was then placed under a sonicator tip for 40 seconds using 7 cycles and 40% power. After that, the homogenates were transferred to new Eppendorf tubes on ice before they were centrifuged in a pre-chilled microfuge at 12,000 rpm for 20 minutes at 4°C. After centrifugation, the supernatants were carefully transferred to new tubes and the deposits were discarded along with their tubes. The samples were then stored at -80°C.

2.15.2 Protein quantification

The protein level in each of the samples was measured using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific), following the manufacturer’s instructions. At the beginning, five albumin standards were prepared from 2 mg/ml stock. In order to achieve accurate measurement, the assay was done in duplicate for each of the standards and the samples. The prepared standards were 0, 2, 4, 8 and 10mg/ml diluted in ddH2O. The samples were diluted 1:10. After that, the required amount of BCA working reagent was calculated considering that each sample or standard needed 190μl of working reagent containing 50 parts of solution A (sodium carbonate, sodium bicarbonate, bicinchoninic acid and sodium tartrate in 0.1M sodium hydroxide) and 1 part of solution B (4% cupric sulphate). The reaction was set up in a microplate (Thermo Fisher Scientific) in which each 10μl of the samples or standards were mixed with

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190μl of the prepared BCA working reagent, and the micropate was incubated at 37°C for 30 minutes. The plate was then loaded into an Ultrospec 3000 optic reader (Pharmacia Biotech) and the optical density measured at 750nm. The readings obtained from the different standards were plotted against their protein concentration in order to generate a standard curve that was used to determine the protein concentration of each sample.

2.15.3 Immunoprecipitation

Antibodies were diluted in PBS at a total volume 450 μl. The experiment was designed to include whirlin and blocked whirlin, plus Munc18 as a control antibody. All the antibodies were diluted 1:200 in tubes, except that the blocked whirlin tube also contained a specific peptide (Santa Cruz, sc-49787 P) to block the whirlin antibody. This peptide is the same against which the antibody had originally been raised, and the amount of whirlin peptide was five times the antibody amount in the tube, based on the manufacturer’s instructions. The tubes were incubated with rotation overnight at 4°C, with the expectation that the blocking peptide would be effective. In order to attach the prepared antibodies to beads, 50μl of PBS-washed magnetic beads (Novex; Life Technologies) were added to each tube and the mixture (500μl) was incubated with rotation at RT for 2 hours. This preparation was called antibody-bound beads. In the middle of the incubation time, 350μg of protein from each sample was prepared in a total volume of 450μl of PBS and mixed with 50 μl of PBS-washed magnetic beads before being incubated with rotation at RT for 1 hour. The tubes were then placed on a magnetic stand for about 2 minutes until clear supernatant formed. The supernatants were saved and the beads were discarded. This step was done to remove any unwanted materials, leaving clean protein samples known as pre- cleared protein. After that, the antibody-bound beads (500μl) were mixed with the pre-cleared protein (500μl) and kept rotating overnight at 4°C in order for the antibody-bound beads to bind to the protein of interest. The next day, the tubes were placed on the magnetic stand for about 2 minutes before the flow through/supernatant was removed from each sample and saved. The beads were then washed by mixing 500μl of PBST, putting the tubes in the magnetic stand and then removing the supernatant. The beads were then resuspended in 30μl

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of 4X loading buffer, boiled at 65°C for 5 minutes in order to dissociate from the proteins, and then placed on ice. Subsequently, the beads were placed in ice and 30μl of eluted supernatant was transferred from each sample to a new Eppendorf tube ready for loading on a gel.