Validation of Ubc9 expression by Western blot and quantitative RT-PCR. Western blot analysis was performed from nuclear extract and cytoplasm extract of K562C/EBPαp30-ER and p42 cell line after β-estradiol induction at different time points using anti-C/EBPα antibody. Western blot analysis was also performed from whole-cell lysates of AML patient cells, K562 C/EBPαp30-ER cells, and C/EBPαp42-ER cells before and after β-estradiol induction at different time points using anti-Ubc9 antibody. (A) Nuclear translocation is induced by treatment with β-estradiol. (B,C) Ubc9 expression in K562C/EBPαp30-ER and K562C/EBPαp42-ER cells after β-estradiol induction. (D) Patients with AML with different subtypes (lanes 1-3, 8, and 9: patients with AML with C/EBPαp30 mutation; lanes 4 and 10: t(8;21); lane 5: Inv(6); lanes 6 and 11: Inv3; and lane 7: normal bone marrow). The numbers underneath the blot are the densitometric values calculated as protein–β-tubulin ratios using ImageJ 1.36 software (National Institutes of Health, Bethesda, MD). (E) Quantitative real-time PCR for the expression of Ubc9 mRNA after β-estradiol induction at different time points. The fold increase for expression was calculated using Δct = (Ct sample − Ct control), and ΔCt values for each sample were standardized by GAPDH Ct value. The fold change was calculated as (= 2^−Δct). Values are expressed as means (± SEM) for 3 independent experiments, with P values shown on histograms.