Figure 1

Non-cleavable poly-Ub in Drosophila. (A) Summary of poly-Ub constructs. (B) Recombinant, untagged Ub⁶ (1 µM) was incubated with USP5 (50 nM). See Methods for details. (C) Western blots of Ub⁶-Stop expressed in all tissues. Driver was sqh-Gal4. HMW: higher molecular weight. Asterisk: non-specific band. Flies were one day old. (D) Western blots from whole flies expressing the indicated transgenes, driven everywhere by sqh-Gal4. HIS⁶-pulldown of Ub⁶-Stop was conducted under denaturing conditions. Solid line separates blots from the same membrane probed with the indicated antibodies. Dotted lines separate lanes reorganized from the same membrane. Un-cropped blots are in Supplemental Figures. (E) Histograms summarizing Ub⁶-Stop that is unmodified (non-conjugated band) vs. modified (conjugated bands and upper smear) in blots in panels (C,F,G) and other, similar experiments. N = 10 independent repeats. Means −/+SD. Driver: sqh-Gal4. (F) Deubiquitination reaction of Ub⁶-Stop isolated from flies and incubated in the absence or presence of the catalytic domain of recombinant USP2 (USP2_CD). See Methods for details. (G) Western blots from denature/renature IPs to examine ubiquitination of HA-tagged Ub⁶-Stop by the indicated linkages. Solid lines indicate that membrane was loaded multiple times with the same samples, same amounts, cut and probed simultaneously, as shown, to eliminate cross-contamination issues from stripping and re-probing the same membrane. Asterisks: non-specific bands that we observe consistently with the respective antibodies. Dotted lines separate lanes cropped from the same membrane. Un-cropped blots are shown in Supplemental Figures. (H) Western blots of whole, adult flies expressing Ub⁶-Stop in all tissues (driver was sqh-Gal4), and loaded to probe all Ub species. HMW: higher molecular weight. Box on the right summarizes quantification of signal from the adjacent blots and other, independent experiments, normalized to respective loading controls, with Ctrl. lanes set to 100%. P value is from two-tailed Student’s t-test. Signal quantified encompassed the band at the arrow all the way at the top of the gel. I) Western blots of stringent purification of HA-Ub⁶-Stop driven by tubulin-Gal4-GS, which is dependent on RU486 to induce expression of Ub⁶-Stop in all tissues; see main text for details. Arrows: unmodified Ub⁶-Stop. Curved lines: higher molecular weight species consistent with ubiquitinated Ub⁶-Stop. For panels (B–D,F–I), results are representative of experiments conducted independently at least thrice, with similar results. In all panels with Drosophila data, flies were heterozygous for driver and Ub⁶.