American Society of Hematology

Figure 5

$Figure 5. Expression and phosphorylation status of C/EBPα in K-562 cells. (A) Expression of C/EBPα transcript. Expressions of C/EBPα transcript in sodium butyrate-treated and untreated K-562 cells (+SB and −, respectively) were compared using real-time PCR. The quantity of the C/EBPα transcript was normalized to that of the β-actin transcript in each sample. Data were obtained from 3 detections; standard deviations are shown. (B) Western blotting for C/EBPα. C/EBPα protein levels in the cytoplasmic and nuclear fractions of sodium butyrate-treated and untreated K-562 cells were analyzed using Western blotting with anti-C/EBPα antibody (1:500 dilution). After stripping, the membrane was detected successively with the antibodies against α-tubulin (1:5000 dilution) and histone H1 (1:500 dilution), which are exclusively present in the cytosol and nucleus, respectively. (C) Phosphorylation status of C/EBPα. Nuclear proteins, prepared from K-562 cells with or without sodium butyrate treatment were immunoprecipitated using anti-C/EBPα antibody. The bound proteins were then analyzed with Western blotting using the following antibodies: anti-phosphoTyr/Thr/Ser (1:4000 dilution), anti-phosphoTyr (1:2000 dilution), anti-phosphoThr (1:1000 dilution), and anti-phosphoSer (1:2000 dilution) (top row of panels). Each blot was then stripped and detected with anti-C/EBPα antibody (bottom row of panels).$

Expression and phosphorylation status of C/EBPα in K-562 cells. (A) Expression of C/EBPα transcript. Expressions of C/EBPα transcript in sodium butyrate-treated and untreated K-562 cells (+SB and −, respectively) were compared using real-time PCR. The quantity of the C/EBPα transcript was normalized to that of the β-actin transcript in each sample. Data were obtained from 3 detections; standard deviations are shown. (B) Western blotting for C/EBPα. C/EBPα protein levels in the cytoplasmic and nuclear fractions of sodium butyrate-treated and untreated K-562 cells were analyzed using Western blotting with anti-C/EBPα antibody (1:500 dilution). After stripping, the membrane was detected successively with the antibodies against α-tubulin (1:5000 dilution) and histone H1 (1:500 dilution), which are exclusively present in the cytosol and nucleus, respectively. (C) Phosphorylation status of C/EBPα. Nuclear proteins, prepared from K-562 cells with or without sodium butyrate treatment were immunoprecipitated using anti-C/EBPα antibody. The bound proteins were then analyzed with Western blotting using the following antibodies: anti-phosphoTyr/Thr/Ser (1:4000 dilution), anti-phosphoTyr (1:2000 dilution), anti-phosphoThr (1:1000 dilution), and anti-phosphoSer (1:2000 dilution) (top row of panels). Each blot was then stripped and detected with anti-C/EBPα antibody (bottom row of panels).

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