FIGURE 2. Eps8 is a substrate of SCF^Fbxw5 in vitro.

(a) In vitro ubiquitylation reaction of His-Eps8 (0.2 μM) with 75 μM His-Ubiquitin, 170 nM Ube1, 1 μM UbcH5b, and 5 mM ATP in the absence or presence of different amounts of control (= flag-IPs from non-transfected cells), flag-Fbxw5, and flag-Fbxw5ΔFbox immunoprecipitates at 30°C for 120 min.

(b) For SCF^Fbxw5 reconstitution, mouse Fbxw5/Skp1 complexes were purified from bacteria (see material and methods) and Cul1/Rbx1 complexes were purified from E.coli using a ”split and co-express“ approach and in vitro neddylated as previously described^{31, 32}. For complex formation, both sub-complexes were mixed in equimolar amounts and incubated on ice for 20 min.

(c) Time course of SCF^Fbxw5-dependent Eps8 ubiquitylation. 0.2 μM His-Eps8 was incubated with 75 μM His-Ubiquitin, 170 nM Ube1, 0.5 μM UbcH5b, 50 nM SCF^Fbxw5, and 5 mM ATP at 30°C. Reactions were stopped at indicated time points, and analyzed by immunoblotting. # stacking gel

(d) In vitro ubiquitylation of His-Eps8 (0.2 μM) with 75 μM His-Ubiquitin, 170 nM Ube1, 1 μM UbcH5b or Cdc34, 5 mM ATP in the absence and presence of 150 nM reconstituted SCF^Fbxw5 at 30°C for 90 min.