I would like to test whether the stability of my protein of interest is increased upon inhibition of proteasomal degradation. I transiently transfect HEK293 cells with my protein of interest (a serine/threonine kinase). I found different concentrations and incubation times (ranging from 1 to 25umol and 4 to 16 hours) and I do not know which concentration and incubation time makes the most sense to answer my question. Additionally, do you suggest to starve the cells prior to adding MG132? My protein of interest is not known to be involved in cell cycle regulation.
I typically use 10 uM for 8 hours in dopaminergic MN9D cells but of course this is another cell type. My suggestion would be to make a dose-response and time curve. For example, use 10 uM for 0, 2, 4, 8 hours. Then if you see an effect at for instance 4 hours, you can make a dose-response curve to determine the optimal concentration. Be aware that blocking the proteasome will lead to some cell death. Usually I serum starve the cells prior to the treatment with MG132.
I hope this was helpful in any way!
Best,
Edward
Why are you looking to starve the cells? Is that a separate experiment or related to your MG-132 experiment. The reason I am asking is because, starvation particularly for amino acids can induce proteasomal degradation via autophagy. So starvation would probably indtroduce an additional unwanted factor in your experiment. If you are looking for cell-cycle synchronization by serum starvation or amino acid starvation, it may be a good idea to do it well before the addition of MG-132 to the cells.
Thank you for your answer! Yes, I intended to achieve cell-cylce synchronization by starvation. At what time point before addition of MG-132 would you suggest to starve the cells?
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