Figure 1.

E6AP inhibits C/EBPα steady state levels. (a) HEK 293T cells were transfected with C/EBPα (0.5 μg) along with increasing amounts of E6AP (0.5–2.0 μg). This was followed by immunoblotting with C/EBPα, E6AP and β-actin antibodies. (b) 293T cells were transfected with C/EBPα (0.5 μg) along with increasing concentrations of E6AP-C843A (0.5–2.0 μg) and were followed by immunoblotting with C/EBPα, E6AP and β-actin antibodies. (c) 293T cells were transfected with C/EBPα (0.5 μg), E6AP (2.0 μg) and E6AP mutant C843A (2.0 μg) as indicated. Post 24 h nuclear extracts were prepared, resolved on 10% SDS-PAGE and probed with C/EBPα, E6AP and GAPDH antibodies. GAPDH was used a control for cytoplasmic protein extract. (d, e) K562 cells were transfected with C/EBPα (0.5 μg), E6AP (1.0, 2.0 μg) and E6AP-C843A (1.0, 2.0 μg). In the indicated condition, cells were treated with 25 μM MG132 3 h prior to cell lysate preparation. The blot was probed with C/EBPα, E6AP and β-actin antibodies. (f) 32Dcl3 cells were transfected with C/EBPα (0.5 μg), E6AP (0.5, 1.0, 1.5 and 2.0 μg) and E6AP-C843A (1.0, 2.0 μg). The blot was probed with C/EBPα, E6AP and β-actin antibodies. (g) 293T cells were transfected with p42C/EBPα (0.5μg), p30C/EBPα (0.5 μg) and E6AP (1.0–2.0 μg) as indicated. Cells were treated with 25 μM MG132 3 h prior to lysate preparation in one of the conditions. Lysates were resolved on 10% SDS-PAGE and probed with C/EBPα, E6AP and β-actin antibodies