Figure 3: UbS2 of ataxin-3 regulates its degradation in cells.

(a) Top: western blots of whole-cell lysates. HeLa cells were transfected as indicated and harvested 24 h later. Bottom: means of ataxin-3 signal quantified from blots on the top and other similar experiments. Error bars: s.d. (b) Quantitative reverse transcriptase-PCR of HeLa cells expressing the indicated constructs. Endogenous control: GAPDH. N=3 independently conducted experiments. Shown are mean ataxin-3 mRNA levels ±s.d. (c,d) Top: western blots of whole-cell lysates of HeLa cells transfected as indicated, treated or not with MG132 (4 h, 15 μM) 48 h later and harvested. Bottom: means of ataxin-3 signal quantified from blots on the top and other similar experiments. Error bars: s.d. (e,f) Top: HeLa cells were transfected as indicated and 24 h later were treated with CHX for the specified amounts of time. For panel e, to have comparable protein amounts of both ataxin-3 versions at time 0 min, we transfected three times more K-null-UbS2* construct than K-null, which was supplemented with empty vector to equate total DNA per group. Western blots of whole-cell lysates. Bottom: means of ataxin-3 signal quantified from blots on the top and other independent experiments. Error bars: s.d. (g) Top: western blots of whole-cell lysates. Two different mutations were used to disrupt UbS2: W87A and W87K, with similar results. Bottom: means of ataxin-3 signal quantified from blots on the top and other independent experiments. Error bars: s.d. (h) Top: HeLa cells were transfected as indicated and 24 h later were treated with CHX for the specified time points. Three times more ataxin-3-UbS2* construct was transfected than ataxin-3-WT to have comparable protein levels at time 0 min. Bottom: means of ataxin-3 signal quantified from blots on the top and other independent experiments. Error bars: s.d. For panels a–d and g, P-values are from analysis of variance with Tukey’s post hoc correction comparing the various versions of ataxin-3 with their panel controls. For panels e,f and h, P-values are from Student’s t-tests. N of independently repeated experiments is specified in panels.