control of proteasome inhibition

paganmar paganmar at tariffenet.it
Mon Jun 7 19:56:03 EST 2004


Hi,
I have been doing experiments in neuro2a cells with inhibitors of
proteasome, like MG132 and lactacystin, because I wanted to show that
the levels of my protein in WB increased upon proteasome inhibition.
Now, since it didn't work, I wonder if the inhibition of the
proteasome itself, for some reason, didn't occur. So, I wanted to
show, as a positive control of proteasome inhibition, that in my
treated lysates the levels of some proteins well known substrates of
proteasome increased (or not ). I tried with cyclin E and p27, but it
didn't work, in part because I serum-starve my cells for other reason,
so I cannot use protein cell-cycle regulated to this purpose. I also
tried with p53, but I suspect the ab they gave me does not recognize
the mouse p53 (n2a is a mouse neuroblastoma cell line). Does anyone
know of any protein which levels dramatically increase upon proteasome
inhibition, which I can use as a positive control in WB? Keep in mind
that I have done time courses 2 through 10 hours, with also a
time-point overnight.
Thanks a lot.
mario



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