Fig. S6.
(A) SUP-36 in vitro ubiquitination assay similar to the assay shown in Fig. 4D, but with longer time points displayed. Each reaction contained T7-SUP-36 and other reaction components as indicated below the gel. All time points are after ATP addition, except lane 1 (0 min). In the presence of CUL-1, UBCH7/HHARI modify SUP-36 with one and two single Ubs (lanes 5–7), whereas no product is detected when CDC34 is the sole E2 (lanes 8–10). All four components (UBCH7/HHARI and CDC34/CUL-1) are required for the formation of poly-Ub chains on SUP-36 (lanes 11–13). (B) Longer time points of the Lys-less Ub assay shown in Fig. 4E. SUP-36 is modified with one or two single Ubs in the presence of all four components. The same pattern is observed when CDC34 is omitted, confirming that CDC34 is required for Ub chain formation (in the presence of WT Ub), and that the multiple SUP-36 bands seen in Fig. 4D (lanes 8–9) and A (lanes 5–7) arise from addition of multiple mono-Ubs. Unmodified SUP-36 and mono- and double-Ub T7-SUP-36 are indicated by arrows. (C) Precharged UBCH7∼Ub was added to reactions containing HHARI, N8-CUL-1, and T7-SUP-36. The time points shown are after UBCH7∼Ub addition. (The 0 s time point was taken immediately before UBCH7∼Ub addition.) The results were visualized by Western blot analysis against T7 (SUP-36). (D) The same assay as in C, but using the SUP-36N109K/F110K mutant at the predicted SUP-36/CUL-1 interface.