Fig. S4.
HHARIRBR does not form poly-Ub chains. Autoubiquitination assays in which the GST-tagged E3 (HHARI) served as a proxy substrate were performed with either WT Ub (Left) or Lys-less Ub (Right). The construct used, GST-HHARIRBR, lacks the Ariadne domain and thus is constitutively active (32). Lys-less Ub is unable to form Ub-chains; therefore, the higher molecular weight bands observed are numerous multiple mono-Ub modifications. Products were visualized by Western blot analysis against the GST-tag on HHARI.