Figure 9. Smurfs are required for ubiquitin modification of Ptch1.
(A) Western analysis of ubiquitinated Ptch1-FLAG and Ptch1Δ2PY-FLAG in Smurf1−/−;Smurf2fl/fl MEFs infected with Ad-GFP or Ad-Cre. These MEFs were first infected with adenoviruses and then transfected with HA-Ub and the Ptch1 plasmids as marked. The exogenously expressed Ptch1 proteins were immunoisolated using anti-FLAG beads prior to analysis. (B) Western analysis of Ptch1-FLAG ubiquitination in vitro in a reconstituted system comprising purified recombinant His6-Smurf2 or the ligase-deficient His6-Smurf2CG from the baculovirus, HA-Ub, and an ATP regeneration system. Ptch1-FLAG was immunoisolated from HEK293 cells and the ubiquitination reaction was carried out on beads. The proteins were eluted prior to Western blot analysis. (C) Western analysis of ubiquitinated Ptch1-FLAG in Smurf2−/− MEFs that were also transfected with Wt, KO, K48, or K63 ubiquitin in the absence or presence of Myc-Smurf2. (D) Western analysis of ubiquitinated Ptch1-FLAG in WT MEFs treated with ShhN or control conditioned medium. Ptch1-FLAG in A-C was resolved by 6% SDS-PAGE, but a 4–12% gradient gel was used in D.