Expression profiles of I antigen and IGnT genes and phosphorylation status of C/EBPα Ser-21 in CD34⁺, CD71⁺, CD15⁺, E-cultured, and G-cultured cells. (A) Flow cytometry analysis for I antigen expression. Adult CD34⁺, CD71⁺, and CD15⁺ cells were purified as described in “Isolation of CD34⁺, CD71⁺, and CD15⁺ cells and E and G cultures.” Flow cytometric analyses demonstrated that more than 95% of the collected cells possessed the respective CD marker. E-cultured and G-cultured cells were obtained through culture of CD34⁺ cells in erythropoietic and granulopoietic conditions, respectively, and more than 99% of the cells became positive for CD71 or CD15 markers after the respective culture procedures. Cell-surface I antigens of these cells were detected with the use of flow cytometry and with monoclonal anti-I antibody. Open and shaded areas represent cells detected with anti-I antibody and FITC-conjugated secondary antibody and with FITC-conjugated secondary antibody only, respectively. The geometric MFI detected is shown on the top of each peak. (B) Expressions of IGnT transcripts. The expressions of IGnT transcripts in the cDNA sample were analyzed with the use of real-time PCR. The quantities of IGnT transcripts were normalized to that of the GAPDH transcript in each sample. Data were obtained from 3 detections; SDs are shown. (C) Expressions of C/EBPα transcripts. The C/EBPα transcript in the cDNA sample was quantitatively analyzed by the use of real-time PCR. The quantities of C/EBPα transcripts were normalized to that of the GAPDH transcript in each sample. Data were obtained from 3 detections; SDs are shown. (D) Western blotting for C/EBPα and C/EBPα with phosphorylated Ser-21. Nuclear fractions of the cells were analyzed by the use of Western blotting with Phospho-C/EBPα(Ser21) antibody. After stripping, the membrane was detected successively with the antibodies against C/EBPα and histone H1.