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closecould this be a b-gal and EGFP coexpression effect?
Posted by derek_japan on 22 Mar 2007 at 07:38 GMT
The Introduction mentions the 2004 paper in which coexpression of b-gal and EGFP was associated with reduced growth and premature lethality in mice [5]. That paper seems to conclude that this was specific for the b-gal/EGFP combination: defects were not observed when EGFP was expressed in the absence of b-gal. Moreover, aggregations of b-gal and EGFP were observed that colocalised with ubiquitin...
From what I understand in the current paper, transfections were all carried out together with b-gal in addition to the various constructs. I wonder if the inhibition observed here are due to the combination of b-gal with EGFP, such that something wierd is going on with ubiquitination when b-gal and EGFP are both expressed together?
Is it possible to do these experiments in the absence of b-gal? Did the in vitro experiment in Fig. 3E where EGFP didn't affect ubiquitination also contain b-gal protein? I wonder if the culprit is really b-gal and not EGFP??
RE: could this be a b-gal and EGFP coexpression effect?
mathijs replied to derek_japan on 11 Apr 2007 at 09:19 GMT
We tested EGFP alone or in combination with bGAl in 293T cells and saw the seem effect on ubiquitination and NF-kB signaling.
And furthermore, the experiments in the cell lines with stable expression of EGFP and in EGFP transgenic mice were also in the absence of bGal. So EGFP seems to be the culprit.