C/EBPαp30 enhances the sumoylation of C/EBPαp42 at K161, leading to its reduced transcriptional activity. His-tagged proteins purified using nickel Ni²⁺-NTA resin. (A) Immunoblot for the eluted proteins using anti-Flag M2 (top panel), anti-C/EBPα (middle panel), and Anti-His₆ (bottom panel) antibodies. The arrows indicate the position of SUMO-1–modified proteins. (B) Transient transfection assay performed in 293T cells with a reporter construct of a minimal TK promoter with CEBP binding sites and expression plasmids for His C/EBPαp42 (WT), C/EBPαp30, and Flag–SUMO-1. (C) His C/EBPαp42 K161R (Mut), His C/EBPαp42 (WT), and C/EBPαp30. pTK (without CEBP sites) was used as control. Luciferase activities were measured 24 hours after transfection, and the values were normalized by using Renilla luciferase PRL-null. Values are expressed as means (± SEM) for 3 to 5 independent experiments, with P value shown as histograms.