Figure 4

Ub⁶-Stop turnover in Drosophila. (A) Left: Western blots of whole fly lysates expressing Ub⁶-Stop via tubulin-Gal4-GS. Flies were reared in media without RU486. On day 1 as adults, they were switched to media with the inducer and allowed to produce Ub⁶-Stop for seven days. On day 7, flies were switched to media without RU486 to shut off Ub⁶-Stop production and whole flies were frozen on the indicated days. Right: quantification of the entire Ub⁶-Stop smear from the images on the left and other, independent experiments. Means −/+SD. N = 6 independent repeats. Two different exposures are shown for Ub signal. (B–D) Western blots from dissected fly heads expressing Ub⁶-Stop and the indicated RNAi lines. Numbers denote different RNAi lines. In all panels, flies were heterozygous for GMR-Gal4, Ub⁶-Stop and RNAi transgenes. Results are representative of experiments conducted independently three or more times with each RNAi line, with similar results. For Rad23 lines we are including results from two independent experiments, since the results are not as clear or consistent as with the other lines from experiment to experiment. Samples, gels and probes were conducted on different days. Samples in one membrane were collected and prepared on the same day. (E) Table summarizing quantification of images from blots in panels (B–D) and other, independent experiments. The entire Ub⁶-Stop signal was quantified. N is at least three for each RNAi line and controls. Signal from RNAi lines was normalized to its respective control. Means −/+SD. P values are from two-tailed Student’s t-tests comparing each RNAi to its own control (no RNAi). Asterisks: p < 0.05.