Protein manipulation

a)Radio-labelling

(i)Samples preparation

A subconfluent monolayer of BHK-21 cells in 6-well plate was infected at an MOI of 1. WT or recombinant viruses, were diluted in PBS – 2% NCS. After adsorption of viruses in 200 µl, plates were incubated at 37˚C and rocked every 20 min. After 1h, 2 ml of appropriate growth media was added and plates were incubated at 33˚C.

At different times post-infection, cells were washed with DMEM methionine-free media,

then replaced with 500 µl of DMEM methionine free containing 35 µCi of TRAN35S-Label.

Plates were incubated for 2h at 37°C to allow labelling of newly synthesized proteins. During the course of the experiment, radioactivity was monitored.

Labelling medium was removed prior to cell lysis. 300 µl of protein disruption buffer was added. Cells were rocked for 10min at RT until the lysate became liquid. Samples were collected in 1.5ml Eppendorf tubes and store and -20°C before use.

Chapter 2: Materials and methods Methods

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(ii)SDS-PAGE gel

Lysates were boiled at 100°C for 3 min, cooled on ice and 10 to 15 µl were loaded into each well of a NuPAGE 4/12% Bis-Tris gel 1.0mmx12wells (Invitrogen). Electrophoresis was carried in 1X MES SDS running buffer at 160V until the Bromophenol Blue reached the bottom of the gel.

(iii)Fixation and drying

The gel was transferred in a fixing solution (50% methanol, 10% acetic acid, 40% H2O) for 10 to 15 min. The gel was rinsed twice in water and then placed into a solution of water with 5% glycerol (v/v) for 5 min. Whatmann paper was soaked with the glycerol solution before the gel was placed onto it. The wetted whatmann paper/gel was covered with cling film and placed into a gel dryer. The drying process was carried on at 80°C for 2h in a vacuum system.

(iv)Detection

The dry gel was inserted in a cassette and the exposure was taken overnight. The radio print was revealed using a Fujifilm phospho-imager and data analysed with the ImageGauge 4.21 software.

b)

Western blotting

A subconfluent monolayer of BHK-21 cells in 6-well plate was infected with an MOI of 1. WT or recombinant viruses, were diluted in PBS – 2% NCS. After inoculation of viruses, plates were incubated at 37˚C and rocked every 20 min. After 1h, 2 ml of appropriate media was added and plates were incubated at 33˚C.

At different time post-infection, cells were lysed using 300 µl of protein disruption buffer. Cells were rocked for 10min at RT until the lysate became liquid. Samples were collected in 1.5ml Eppendorf tubes and store at -20°C before use.

(ii)SDS-PAGE gel

Lysates were boiled at 100°C for 3 min, left to cool down and 10 to 15 µl were loaded into each well of a NuPAGE 4/12% Bis-Tris gel 1.0mmx12wells. Electrophoresis was carried out in 1X MES SDS running buffer at 160V until the BPB reached the bottom of the gel.

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(iii)Electro blotting

The transfer of the proteins from the gel to a nitrocellulose membrane was performed using a semi-dry transfer apparatus (Bio-Rad Trans-Blot SD). Beforehand, the gel, two pieces of Watmann paper and the nitrocellulose membrane (Hybond C extra) cut to the size of the gel were rinsed in transfer buffer.

First, one layer of Watmann paper was place on the blotter, the nitrocellulose membrane was placed on top of it. Then, the gel was carefully layered on top. A final piece of Watmann paper was placed on top of the stack. Possible bubbles were removed by rolling a pipette on top of the stack.

The apparatus was further assembled by fitting the lid on and transfer was carried out at for 40 min at a constant voltage of 10 V.

(iv)Detection

After transfer, the membrane was taken out of the blotter. Blocking was carried out in a plastic box containing ~30 ml of blocking buffer for 1h at room temperature and under constant agitation.

Binding of primary antibodies was carried out in a 50 ml falcon tube containing 10 ml of blocking solution and the appropriate concentration of anti-BUN N or anti-Tubulin antibodies. Primary antibodies were incubated either for 1h at room temperature or overnight at 4°C, in any case constant rotation was applied.

The membrane was moved back to the plastic box, and washed 3 times in blocking buffer for 5 min. Then, the membrane was incubated with the appropriate secondary antibodies in a new 50 ml falcon tube for 1h at room temperature under constant rotation. The membrane was transferred back into the plastic box and washed 3 times in blocking buffer for 5 min.

The membrane was rinsed in PBS – 0.1% Tween 20. Before placing the membrane on a plastic film, the remaining PBS was drained. Then, an enhanced chemiluscent substrate was added to the membrane. After 1 min of incubation, the membrane was transferred onto a new piece of plastic and placed into a cassette.

X-ray films were used to visualise the light signal. X-ray films were exposed from 1 sec to 25 min according to the signal strength and reveal using Kodak automatic developer.

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