Figure 6
Figure 6. C/EBPα phosphorylation is regulated by PI3K/PKB. (A) Cells were starved overnight in absence of cytokines and in presence of 0.5% FCS. Cells were left untreated (lane 1) or treated with 10 μM LY294002 (lane 2), 20 μM HIMO (lane 3), and 10 μM SB216763 (lane 4) for 1 hour before protein lysates were made. Western blot analysis was performed with an antibody against phosphorylated C/EBPα, total C/EBPα, and as a control for equal loading an antibody against β-actin. (B) CD34+ cells were retrovirally transduced with eGFP as a control (lane 1) or PKBcaax (lane 2). After 7 days of differentiation in presence of IL-3 and IL-5 to induce eosinophil differentiation, transduced cells were separated from the nontransduced cells by FACS, and protein lysates were made. Western blot analysis was performed with an antibody against phosphorylated C/EBPα (Τ221/226) and an antibody against β-actin. Vertical line(s) have been inserted to indicate a repositioned gel lane. (C) CD34+ cells were retrovirally transduced with eGFP as a control (lane 1) or myrPKB (lane 2). After 7 days of differentiation in presence of IL-3 and IL-5 to induce eosinophil differentiation, transduced cells were separated from the nontransduced cells by FACS, and protein lysates were made. Western blot analysis was performed with an antibody against phosphorylated JunB and an antibody against β-actin (D).CD34+ cells were retrovirally transduced with eGFP as a control (lane 1) and constitutively active GSK-3 (lane 2). After 7 days of differentiation in presence of IL-3 and IL-5 to induce eosinophil differentiation, transduced cells were separated from the nontransduced cells by FACS, and protein lysates were made. Western blot analysis was performed with an antibody against phosphorylated C/EBPα (Τ221/226), JunB, and an antibody against β-actin. (E) CD34+ cells were retrovirally transduced with eGFP (lane 1) or C/EBPαT222/226A (lane 2). After 7 days of differentiation in presence of IL-3 and IL-5 to induce eosinophil differentiation, transduced cells were separated from the nontransduced cells by FACS, and protein lysates were made. Western blot analysis was performed with an antibody against C/EBPα (Τ221/226), C/EBPα JunB, and an antibody against β-actin. Vertical line(s) have been inserted to indicate a repositioned gel lane.

C/EBPα phosphorylation is regulated by PI3K/PKB. (A) Cells were starved overnight in absence of cytokines and in presence of 0.5% FCS. Cells were left untreated (lane 1) or treated with 10 μM LY294002 (lane 2), 20 μM HIMO (lane 3), and 10 μM SB216763 (lane 4) for 1 hour before protein lysates were made. Western blot analysis was performed with an antibody against phosphorylated C/EBPα, total C/EBPα, and as a control for equal loading an antibody against β-actin. (B) CD34+ cells were retrovirally transduced with eGFP as a control (lane 1) or PKBcaax (lane 2). After 7 days of differentiation in presence of IL-3 and IL-5 to induce eosinophil differentiation, transduced cells were separated from the nontransduced cells by FACS, and protein lysates were made. Western blot analysis was performed with an antibody against phosphorylated C/EBPα (Τ221/226) and an antibody against β-actin. Vertical line(s) have been inserted to indicate a repositioned gel lane. (C) CD34+ cells were retrovirally transduced with eGFP as a control (lane 1) or myrPKB (lane 2). After 7 days of differentiation in presence of IL-3 and IL-5 to induce eosinophil differentiation, transduced cells were separated from the nontransduced cells by FACS, and protein lysates were made. Western blot analysis was performed with an antibody against phosphorylated JunB and an antibody against β-actin (D).CD34+ cells were retrovirally transduced with eGFP as a control (lane 1) and constitutively active GSK-3 (lane 2). After 7 days of differentiation in presence of IL-3 and IL-5 to induce eosinophil differentiation, transduced cells were separated from the nontransduced cells by FACS, and protein lysates were made. Western blot analysis was performed with an antibody against phosphorylated C/EBPα (Τ221/226), JunB, and an antibody against β-actin. (E) CD34+ cells were retrovirally transduced with eGFP (lane 1) or C/EBPαT222/226A (lane 2). After 7 days of differentiation in presence of IL-3 and IL-5 to induce eosinophil differentiation, transduced cells were separated from the nontransduced cells by FACS, and protein lysates were made. Western blot analysis was performed with an antibody against C/EBPα (Τ221/226), C/EBPα JunB, and an antibody against β-actin. Vertical line(s) have been inserted to indicate a repositioned gel lane.

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