Figure 3

WWOX inhibits ITCH-mediated ubiquitination of ΔNp63 and increases its half-life. (a) HEK293 cells were transfected with the indicated plasmids. After 24 h, cells were treated with 20 μmol/l MG132 for 4 h. Lysate was prepared and IP with anti-Myc (ΔNp63α) and detected with anti-HA antibodies (UB). (b) HEK293 cells were cotransfected with plasmids encoding HA–ΔNp63α and Flag–ITCH and Myc–WWOX or Myc–WWOX-Y33R. After 24 h, cells were lysed and immunoprecipitation was performed as follows: lanes 2 and 7: anti-HA; lanes 3 and 8: anti-IgG; lanes 4 and 9: anti-Myc; and lanes 5 and 10: anti-Flag antibodies. Immunoblotting was done using anti-HA–HRP, anti-Myc_HRP, or anti-Flag-HRP antibodies. Lanes 1 and 6 show 2.5% of input of each lysate. A higher band in the IgG (lanes #3 and 8) and Flag (lanes #5 and 10) is observed in the anti-Myc blot (middle) likely due to antibody non-specificity. (c) HEK293 cells were cotransfected with WWOX and ΔNp63α. After 24 h, cells were treated with 20 μg/ml CHX at the indicated time points and analyzed as shown. (d) Tet-On-inducible SaOS2 cells were infected with 10 MOI of Ad5-WWOX for 24 h then induced with doxycycline for additional 24. Cells were treated with CHX as indicated and blotted with anti-HA or anti-WWOX. Vinculin was used as loading control. (e) HaCaT cells were transduced with lentiviral-vector of WWOX (KD) or scramble shRNA (EV) constructs. Lysates were analyzed using the indicated antibodies. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (f) Tet-On-inducible SaOS2 cells were used as in (d) but instead of CHX, cells were treated with the proteasome inhibitor MG132