American Society of Hematology

Figure 5

$Figure 5. Expressions of I antigen and IGnT genes and phosphorylation status of C/EBPα Ser-21 in erythroid differentiation of K-562 cells. K-562 cells were cultured in medium supplemented with 2mM sodium butyrate (SB) for 2 days or 25μM hemin for 4 days to induce erythroid differentiation. (A) Flow cytometric analysis for I antigen expression detected with monoclonal anti-I antibody. Open and shaded areas represent cells detected with anti-I antibody and FITC-conjugated secondary antibody and with FITC-conjugated secondary antibody only, respectively. The geometric MFI detected is shown on the top of each peak. (B) Expression profiles for the IGnT transcripts. Real-time PCR was used to quantify the IGnTA, IGnTB, IGnTC, and GAPDH transcripts in the cDNA samples. The quantities of the IGnT transcripts were normalized to that of GAPDH transcript in each sample. Data were obtained from 3 detections; SD are shown. (C) Expression of C/EBPα transcript. Expressions of C/EBPα transcript in the cDNA samples were analyzed by the use of real-time PCR. The quantity of the C/EBPα transcript was normalized to that of the GAPDH transcript in each sample. Data were obtained from 3 detections; SD are shown. (D) Western blotting for C/EBPα and C/EBPα with phosphorylated Ser21. Nuclear fractions of the cells were analyzed using Western blotting with Phospho-C/EBPα(Ser21) antibody (1:1000 dilution), which specifically detected C/EBPα with phosphorylated Ser21 residue. After stripping, the membrane was detected successively with antibodies against C/EBPα (1:500 dilution) and histone H1 (1:500 dilution). The results were analyzed by densitometry, and quantification showed that the ratio of C/EBPα with phosphorylated Ser-21 in mock, SB-treated, and hemin-treated cells, after calibration with individual amounts of total C/EBPα and loading control histone, was 1.00:0.36:0.28.$

Expressions of I antigen and IGnT genes and phosphorylation status of C/EBPα Ser-21 in erythroid differentiation of K-562 cells. K-562 cells were cultured in medium supplemented with 2mM sodium butyrate (SB) for 2 days or 25μM hemin for 4 days to induce erythroid differentiation. (A) Flow cytometric analysis for I antigen expression detected 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) Expression profiles for the IGnT transcripts. Real-time PCR was used to quantify the IGnTA, IGnTB, IGnTC, and GAPDH transcripts in the cDNA samples. The quantities of the IGnT transcripts were normalized to that of GAPDH transcript in each sample. Data were obtained from 3 detections; SD are shown. (C) Expression of C/EBPα transcript. Expressions of C/EBPα transcript in the cDNA samples were analyzed by the use of real-time PCR. The quantity of the C/EBPα transcript was normalized to that of the GAPDH transcript in each sample. Data were obtained from 3 detections; SD are shown. (D) Western blotting for C/EBPα and C/EBPα with phosphorylated Ser21. Nuclear fractions of the cells were analyzed using Western blotting with Phospho-C/EBPα(Ser21) antibody (1:1000 dilution), which specifically detected C/EBPα with phosphorylated Ser21 residue. After stripping, the membrane was detected successively with antibodies against C/EBPα (1:500 dilution) and histone H1 (1:500 dilution). The results were analyzed by densitometry, and quantification showed that the ratio of C/EBPα with phosphorylated Ser-21 in mock, SB-treated, and hemin-treated cells, after calibration with individual amounts of total C/EBPα and loading control histone, was 1.00:0.36:0.28.

This Feature Is Available To Subscribers Only