Itch, Smurf2, and NEDD4 polyubiquitinate Glis3.

<p>A-B. HEK293T cells were transfected with CMV-HA-Ubiquitin, FLAG-Glis3 or FLAG-Glis3-ΔC480 or their respective <i>PY</i>^<i>461</i> mutants, and Myc-Itch, Smurf2, NEDD4, or empty vector as indicated. Cells were treated with 10 μM MG132 for 6 h prior to harvest. Co-immunoprecipitation was performed using an anti-M2 FLAG antibody and immunoprecipitated proteins were analysed by Western blot using a high affinity anti-HA, anti-M2 FLAG-HRP, anti-Myc or goat anti-mouse-HRP antibodies. C. HEK293T cells were transfected with FLAG Glis3, CMV-HA-Ubiquitin, and Myc-Itch or its catalytically inactive mutant as indicated. Co-IP was performed as described in A-B. D. HEK293T cells were transfected with FLAG-Glis3, Myc Itch, and HA-Ubiquitin or the K48R or K63R ubiquitin mutants as indicated. Co-IP was performed as described in A-B.