FIGURE 3.

In vitro ubiquitination of Axin by Smurf2. A, S·His-Axin or S·His (as a control; lane 2) was incubated at 37 °C for 2 h with or without purified ubiquitin, E1, E2 (UbcH5C), and E3 (Smurf2 or Smurf2(C716G)) as indicated. The reaction samples were subjected to pull-down analysis using S-protein-agarose, and polyubiquitinated Axin was detected by immunoblotting with anti-ubiquitin antibody. WB, Western blot. B, [³⁵S]Met-labeled Axin was incubated with the components indicated. Polyubiquitinated [³⁵S]Met-labeled Axin was detected by autoradiography. C, S·His-Axin-(1–686) was incubated in the presence or absence of a purified mutant form of ubiquitin whose seven lysine residues were changed to arginine, E1, E2 (UbcH5C), and Smurf2 for 2 h at 37 °C. The reaction samples were pulled down using S-protein-agarose, resolved by SDS-PAGE, and stained with Coomassie Brilliant Blue. The areas marked with rectangles were excised, and MS/MS spectrometric analysis was performed as described under “Experimental Procedures.” D and E, shown are MS/MS spectra for Axin and mono-ubiquitinated Axin, respectively. F, Myc-Axin or Myc-Axin(K505R) with HA-ubiquitin and Myc-EGFP were cotransfected with or without FLAG-Smurf2 into HEK293T cells, followed by immunoblotting.