Figure 2
From: Expression and Regulation of Deubiquitinase-Resistant, Unanchored Ubiquitin Chains in Drosophila
Ub6-Stop does not inhibit protein degradation or affect proteasome levels in Drosophila. (A) Western blots from cytoplasmic/nuclear fractionation of whole flies expressing Ub6-Stop in all tissues (Methods). Results are representative of experiments conducted independently at least three times, with similar results. Asterisks: non-specific signal. Driver was sqh-Gal4. Two different exposures are shown for Ub signal. (B,C) Western blots of the levels of the indicated proteins in the absence or presence of Ub6-Stop. Boxes underneath blots show quantification summaries of the levels of the respective proteins. Means −/+SD. Statistics: two-tailed Student’s t-tests. For (B) CL1-GFP was co-expressed alongside Ub6-Stop in fly eyes, driven by GMR-Gal4. Flies were one day old. Full blots are in Supplemental Figures. For (C) we probed one day old whole adults expressing Ub6-Stop in all tissues through sqh-Gal4. The different lanes in blots represent independent experimental repeats. (D) Levels of endogenous proteasome subunits when Ub6-Stop is expressed everywhere via sqh-Gal4. Flies were one day old. Boxes underneath blots show quantification summaries of the levels of the respective proteasome proteins. Means −/+SD. Statistics: two-tailed Student’s t-tests. The different lanes in all blots represent independent experimental repeats. Tubulin signal in top blot remains after stripping to probe with the antibodies indicated in that panel. In all panels, flies were heterozygous for driver and transgenes.
