Figure 3.

E6AP targets C/EBPα for ubiquitin proteasome pathway and negatively affects its transactivation capacity. (a) 293T cells were transfected with C/EBPα (0.5 μg) and E6AP (2.0 μg); cells were treated with 25 μM MG132 3 h prior to cell harvesting. Lysates were resolved on 10% SDS-PAGE and probed with C/EBPα, E6AP and β-actin antibodies after stripping and reprobing the same blot. (b) 293T cells were transfected with expression plasmids for C/EBPα-HA (1.0 μg), E6AP (2.0 μg), E6AP-C843A (2.0 μg) and ubiquitin-His (1.0 μg) as indicated. Twenty four hours post transfection, cells were treated with 25 μM MG132 3 h prior to harvesting in the indicated lanes. C/EBPα was co-immunoprecipitated and probed with His antibody. The same membrane was stripped and probed with HA antibody (lower panel). (c) 293T cells were transiently transfected with pTK-C/EBP-luc reporter and expression plasmids for C/EBPα, E6AP and E6AP-C843A. Twenty four hours post transfection, luciferase activity was measured. 25 μM MG132 treatment was given 3 h prior to cell harvesting for luciferase activity measurement. Data are representative of three independent experiments. Results are given as standard error of mean (±S.E.M.); *P<0.05; **P<0.001, ***P<0.0001