Figure 4: UIMs of ataxin-3 oppose the effect of UbS2 mutation.
From: Ubiquitin-binding site 2 of ataxin-3 prevents its proteasomal degradation by interacting with Rad23
(a) Top: HeLa cells were transfected as indicated and harvested 48 h later. Western blots from whole-cell lysates. Bottom: means of ataxin-3 signal quantified from blots on the top and other similar experiments. P-values are from analysis of variance (ANOVA) with Tukey’s post hoc correction comparing K-null ataxin-3 with UbS2 mutated, and K-null ataxin-3 with UbS2 and UIMs mutated to K-null ataxin-3 with intact domains. Error bars: s.d. N=10 independently conducted experiments. (b) Top: HeLa cells were transfected with the indicated constructs. Three times more UbS2* DNA was used than UbS2*-UIM* to begin with approximately the same amount of protein at time 0 h. CHX was added to cells 24 h post transfection for the specified time points. Western blots of whole-cell lysates. Bottom: means of ataxin-3 signal quantified from blots on the top and other similar experiments. P-values are from Student’s t-tests comparing ataxin-3 with UbS2 mutated with ataxin-3 with UbS2 and UIMs mutated. Error bars: s.d. N=6 independently conducted experiments. (c) Top: HeLa cells were transfected with the indicated short interfering RNA (siRNA) constructs to knock down endogenous Rad23A, endogenous Rad23B or both, and harvested 48 h later. Shown are western blots of whole-cell lysates. siRNA control: scramble controls. Bottom: means of ataxin-3 signal quantified from blots on the top and other similar experiments. P-values of <0.01 are indicated by ‘**’, and are from ANOVA/Tukey comparing the levels of ataxin-3 protein in RNAi lanes with those in scramble control. Error bars: s.d. N=7 independently conducted experiments.
