Figure 6

Ub⁶-GG is conjugated to proteins in mammalian cells and in flies. (A) HEK-293 cells were transiently transfected as indicated and harvested for whole lysate western blotting 24 hours later. No Ub⁶: empty vector control transfection. 1 × and 2 × denote relative transfection amounts. (B) HEK-293 cells were transfected with the empty host vector of Ub⁶-GG or with HA-tagged Ub⁶-GG for 24 hours. Cells were pelleted, lysed in RIPA buffer with protease inhibitors (PI) and subjected to a denature/renature protocol (Methods) before IP. Asterisks: non-specific signal. Red arrows: unmodified, full length ataxin-3 protein. Ataxin-3 runs as a doublet in input blots. This reflects the two alleles of the gene that have different CAG repeats. Dotted lines separate lanes from the same membrane, cropped and reorganized for ease of viewing. Un-organized blots are in Supplemental Figures. (C) Longevity from adult flies expressing, or not, Ub⁶-GG in all tissues via sqh-Gal4. P value was calculated using log-rank (Mantel-Cox) test. (D) Western blots from denature/renature HIS⁶-based precipitation of Ub⁶-GG expressed in all neurons alongside ataxin-3. Ctrl: no HIS⁶-Ub⁶-GG expressed alongside ataxin-3. Membrane was loaded with the same samples twice, cut in half and probed simultaneously with the indicated antibodies. We used a pan-Ub antibody in this case to show the specificity of the pulldown. Asterisk: non-specific band in the absence of HIS⁶-Ub⁶-GG expression in the fly. Red arrow: unmodified ataxin-3 protein. Blue arrow: ataxin-3 protein fragment most likely resulting from proteolytic cleavage⁷⁷; this species of ataxin-3 is absent in IP lanes. Two different exposures are shown for anti-ataxin-3 signal. Dotted line separates lanes from the same membrane, cropped and reorganized for ease of viewing. Un-organized blots are in Supplemental Figures. For all panels, results are representative of experiments conducted independently at least three times, with similar results.