Figure 1

WWOX physically interacts with ΔNp63α but not TAp63α. (a) HEK293 cells were cotransfected with plasmids encoding Myc–WWOX and HA–TAp63α or HA–ΔNp63α. After 24 h, cells were lysed and immunoprecipitation was performed as follows: lanes 2 and 5: anti-HA; lanes 3 and 6: anti-IgG; and lanes 4 and 7: anti-Myc antibodies. Immunoblotting was done using anti-HA–HRP or anti-Myc-HRP antibodies. Lane 1 shows input of TAp63α and lane 8 shows input of ΔNp63α. (b) SaOS-2 cells, stably transfected with a doxycycline-inducible vector containing the HA–TAp63α or the HA–ΔNp63α gene, were infected with 10 MOI of Ad5-WWOX for 24 h then induced with doxycycline for an additional 24 h. Subsequently, cells were lysed and immunoprecipitation was performed as follows: lanes 1 and 4: anti-HA; lanes 2 and 5: anti-IgG; and lanes 3 and 6: anti-WWOX antibodies. Immunoblotting was done using anti-HA–HRP and anti-WWOX antibodies. (c) HEK293 cells were transfected with plasmids encoding HA–TAp63α or HA–ΔNp63α. After 24 h, cells were lysed and GST-pulldown was performed using GST alone (lanes 2 and 5) or GST–WWOX (lanes 3 and 6) or GST–WWOX-Y33R (lanes 4 and 7). Lanes 1 and 8 show 2.5% of input of each lysate. (d) HEK293 cells were cotransfected with plasmids encoding HA–ΔNp63α and Myc–WWOX. After 24 h, cells were lysed and immunoprecipitation was performed as follows: lane 2: anti-HA; lane 3: anti-IgG; and lane 4: anti-Myc antibodies. Immunoblotting was done using anti-p63 and anti-WWOX antibodies. Lane 1 shows 2.5% of input of lysate. A higher band in the IgG (lane #2) and Myc (lane #3) is observed in the anti-WWOX blot (lower) likely due to antibody non-specificity