the assay lined out is to analyse the expression timecourse of  any recombimant protein in an adherent culture, it describes the assay done in a six well plate, but you can use any format you like. wether you use S9 or High five cells depends on your personal preferences, to make a complete optimization you might wonna analyze both of them.
As a starting point I'll describe the use of a MOI of 5 and 10, check also other MOIs to reach for an optimal expression protocoll, remember that you can't extrapolate from one proteinl to another.
 

1.        Seed two six well plates at a density of 10⁶ cells per well. The final volume of media should be about 1-1,5 ml, this keeps the virus concentrated and faciliates the handing of the supernatant later.

2.        Label plate#1 with MOI 5 and plate #2 with MOI 10 respectivly.

3.        On each plate label wells #1-5 with the timepoints you want to analyze. normally as a start this will be; 24 h, 48 h, 72h, 96 h and 120h. This are the harvesting timepoints, you have only five timepoints, because the remaining wells serve as a uninfected control, label it as one either.

4.        Take one plate and infect the cells at MOI 5 and take the other one and infect it at MOI 10. this means into the MOI 5 you put 5.000.000 pfu of your virus and into the wells with MOI 10 its 10.000.000.

5.        Cover your plates and incubate at 27^oC.

6.        Harvest each well at  the designated timepoint. Take supernatant and cells in a different tube. for the cells I recommend simply to wash them of with 1ml of icecold PBS or to wash them away together with the remaining media, thats what I do..

7.        Pellet the cells at 800xg for ten minutes at 4^oC, keep on ice.

8.        Take of supernatant and transfer it to a seperate tube.

9.        Store the fractions at -70^oC until use.
 

Processing your Timepoint Samples

1.      Thaw all fractions on ice.

2.       Make up the lysis buffer you want to use (e.g. PBS, 0.1% Tx100) Use 100ml per 10⁶ cells, add the protease inhibitors you will use also in the real extraction .
Keep all the stuff on ice all time. Add the lysis buffer to each cell pellet, vortex each cel sample to break up thecells and begin lysis.

3        Incubate the cells 30-45 ' on ice, vortex every now and then to support lysis.

4.        Centrifuge at 1000xg for 10' to pellet cell debris. Transfer the supernatant to a new tube. Analyze the lysate and supernatant by western, use 10 ml of both.