I've treated my ovarian cancer cells with two inhibitors which are cycloheximide (inhibit denovo protein synthesis) and MG-132 (Inhibit proteasome degradation). What I would like to know is what can I use as a positive control to confirm that these inhibitors worked? I'm doing Western Blot with the cell lysates.
Ignore the two comments above, they don't answer your question as far as finding ways to confirm that your inhibitors are working. They unfortunately mistake vehicle control with your request of a "positive control" (or more specifically, relevant readouts for inhibitor activity in your case). In MG-132 treated cells, you should induce a massive accumulation of polyubiquitinated proteins, so a western blot of your cell lysates with anti-ubiquitin +/- MG132 should easily reveals if you indeed blocked proteasomal activity. On the other hand, cycloheximide has been shown to deplete ubiquitin levels in cells (in addition to block protein synthesis, which presumably results in a decrease in steady-state polyubiquitinated proteins). So, using the same approach as with MG132-treated cells, you may be able to detect a decrease in polyubiquitinated proteins by western blot in cells treated with cycloheximide vs untreated. Note that anti-ubiquitin antibodies on the market tend to be messy and careful washing and optimization may be required. Best of luck in your studies! -L
Ideally, one should treat the cells with the vehicle (PBS/DMSO/Ethanol) used to dissolve these inhibitors and use the cell lysates obtained from them as control.
Ignore the two comments above, they don't answer your question as far as finding ways to confirm that your inhibitors are working. They unfortunately mistake vehicle control with your request of a "positive control" (or more specifically, relevant readouts for inhibitor activity in your case). In MG-132 treated cells, you should induce a massive accumulation of polyubiquitinated proteins, so a western blot of your cell lysates with anti-ubiquitin +/- MG132 should easily reveals if you indeed blocked proteasomal activity. On the other hand, cycloheximide has been shown to deplete ubiquitin levels in cells (in addition to block protein synthesis, which presumably results in a decrease in steady-state polyubiquitinated proteins). So, using the same approach as with MG132-treated cells, you may be able to detect a decrease in polyubiquitinated proteins by western blot in cells treated with cycloheximide vs untreated. Note that anti-ubiquitin antibodies on the market tend to be messy and careful washing and optimization may be required. Best of luck in your studies! -L
Alternatively probe your western with cyclin B antibody. If cycloxeximide is working the cyclin levels should go down as they cannot be replenished after anaphase, while in MG132, cyclin levels should increase as it cannot be destroyed by the proteasome
I would suggest beta-catenin, normally this protein is degraded in proteasome, b-cat possess a short half-life so you will be able to see accumulation!
https://en.wikipedia.org/wiki/Beta-catenin
You can also use a p27 anti-body. If you detect a increase in the protein levels, your MG132 worked.
beta catenin is a good read out for MG132 treatment other than ubiquitin. If you lyse your cells directly in lameli buffer then you don't need to add ubiquitinase inhibitors.
You can try to see HIF1a accumulation during normoxia, this protein is only stable during hypoxic exposure...
We would like to make sure that our polyubiquitin Ab is working in cell immunofluorescence (IF). As a positive control can we use MG-132 treated cells? Has anybody tried that by IF?
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