Figure 6.
E3-mediated autoubiquitination with Ub and phosphoUb
- A Time course for a ligase reaction with GST-tagged cIAP1 (aa 363–614), UBE2D1 and Ub or phosphoUb. See Supplementary Fig S11D for a reaction with UBE2D3.
- B Anti-Ub Western blot of an identical reaction run for 3 h.
- C Comparison of the number of Ub chain linkages in the reaction in (B).
- D Ub chain linkage profile for the reaction in (B).
- E Reaction of GST-tagged TRAF6 (aa 1–285) with UBE2D3 as in (A). See Supplementary Fig S11E and F for additional TRAF6 reactions.
- F Anti-Ub Western blot of GST-TRAF6/UBE2D1 run for 3 h.
- G Comparison of the number of Ub chain linkages in the reaction in (F).
- H Linkage composition in GST-TRAF6-UBE2D1 reactions reveals 58% Lys63 chains with wt Ub and UBE2D1. Significantly smaller quantities of linkages are assembled with phosphoUb.
- I-K Chain assembly as in (A) with UBE2L3 and (I) GST-tagged NEDD4L (aa 576–955) (Mund & Pelham, 2009), (J) NleL (aa 170–782) (Hospenthal et al, 2013) and (K) HOIP RBR-LDD (aa 699–1072) (Smit et al, 2012). While the HECT E3 ligases NEDD4L and NleL are active with Ub and phosphoUb, HOIP is significantly less active with phosphoUb. See also Supplementary Fig S11G and H.