Figure 3

Sequestration of the ubiquitin conjugates into insoluble aggregates by Atx3.
(a) Sequestration of overexpressed Ub conjugates. FLAG-tagged Atx322Q-IC, Atx3100Q-IC or Atx3100Q-IC/UIM* was co-transfected with HA-Ub into HEK 293T cells. The cell lysates were subjected to fractionation and Western blotting with an anti-FLAG or anti-HA antibody. (b) Sequestration of endogenous Ub conjugates. FLAG-tagged Atx322Q-IC, Atx3100Q-IC or Atx3100Q-IC/UIM* was transfected into HEK 293T cells. The cell lysates were subjected to fractionation and Western blotting with an antibody against Ub. Sup., supernatant; Pel., pellet. Atx3-IC, C-terminal fragment (residues 221–360) of isoform-I Atx3; UIM*, mutation in UIM domain, S236A/S256A.