Hello all, I created Usp7 shRNA myeloma cell lines using the dox inducible vector pTRIPZ with lentivirus. After multiple attempts I get a robust depletion of Usp7 in the nucleus but the cytoplasm levels of Usp7 do not change much. Total protein levels sometimes show Usp7 depletion but not that strong and other times there is no change at all (however when I fractionate the nucleus is always robustly depleted). Usp7 is a nuclear protein but does this justify this effect? Any thoughts or comments are much appreciated.
Hi Reinaldo,
Maybe this work could help.. http://www.ncbi.nlm.nih.gov/pubmed/15722335?dopt=Abstract
Hi Reinaldo,
And what are the results of qPCR for Usp7 ? Any down regulation?
As far as I know, gene expression down-modulation by RNA interference (shRNA or siRNA) is more effective on cytoplasmic targets, since RISC complex is mainly located in the cytoplasm. Thus, the differential modulation you observe seems quite curious. However, the paper suggested by Marina suggests a possible new mechanism in the selective down-modulation of nuclear mRNAs/proteins. As Andrey also suggests, I would also check the effect on mRNA by qPCR analysis.
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