Ubc9 knockdown prevents sumoylation of C/EBPαp42 and enhances its transcriptional activity in the presence of the dominant-negative C/EBPαp30 isoform. (A) Western blot analysis performed for the expression of Ubc9 in 293T cells at different time points after the overexpression of Ubc9 siRNA and a nonsilencing siRNA control. The numbers underneath the blot are the densitometric values calculated as protein–β-tubulin ratios. (B) A sumoylation assay performed after the overexpression of His C/EBPαp42 (WT), C/EBPαp30, Flag–SUMO-1, siRNA against Ubc9, and control siRNA, and immunoblot for the eluted proteins using anti–Flag M2 (top panel), anti-C/EBPα (middle panel), and anti-His₆ (bottom panel) antibodies. (C) Transient transfection assay performed in K562 cells using the Nucleofector Kit (AMAXA) with the reporter construct of a minimal TK promoter with CEBP-binding sites and expression plasmids for C/EBPαp42, C/EBPαp30, Ubc9 siRNA, and control siRNA. Values are expressed as means (± SEM) for 3 independent experiments, with P values shown on histograms.