Figure 1.
Figure 1. Tet-regulated expression of c-Myb protein in c-myb knockout ES cells. (A) Wild-type ES cells (WT), c-myb–/–parental ES cells expressing tTA (Tet/KO), and c-myb–/–ES cells carrying the Tet-regulated c-myb transgene (c-mybTet/KO) were cultured for 2 days in the presence (1 μg/mL) or absence of Tet. Cells were lysed and subjected to immunoprecipitation and Western blotting with an anti–c-Myb antibody. Two isoforms of the c-Myb protein, p75 (filled arrow) and p89 (open arrow), were detected in wild-type cells, whereas only p75 was detected in c-mybTet/KO cells cultured in the absence of Tet. Total-cell lysates were blotted with antiactin to normalize the initial amount of protein. (B) c-mybTet/KO ES cells were cultured for 2 days in the presence of indicated concentrations of Tet (0-1000 ng/mL). Cells were lysed and subjected to immunoprecipitation and Western blotting as described under “Materials and methods.” (C) c-mybTet/KO ES cells were cultured in the presence (top panels) or absence (bottom panels) of Tet (1 μg/mL) for 2 days. The culture conditions were then shifted to either with Tet (1 μg/mL) (bottom panels) or without Tet (top panels), and incubation continued for another 6 to 24 hours. Cells were lysed and subjected to immunoprecipitation and Western blotting. (D) c-mybTet/KO ES cells were cultured in the presence (top panels) or absence (bottom panels) of Tet (1 μg/mL) for 2 days. The culture conditions were then shifted to either with Tet (1 μg/mL) (bottom panels) or without Tet (top panels), and incubation continued for another 12 to 96 hours. Cells were then analyzed for EGFP expression by flow cytometry. Histograms show intensity of EGFP fluorescence on the cells harvested at indicated time points (green) together with cells that were kept in the presence of Tet as a negative control (black). (E) c-mybTet/KO ES cells were allowed to differentiate on OP9 stromal cell layers in the presence of Tet (1 μg/mL) for 4 days. Flk-1+cells were sorted by FACS and recultured with OP9 cells in the presence (top panel) or absence (bottom panel) of Tet (1 μg/mL). After 24 hours, the culture conditions were shifted to either with Tet (1 μg/mL) (bottom panel) or without Tet (top panel), and incubation continued for another 6 to 24 hours. Cells were then lysed and subjected to immunoprecipitation and Western blotting with an anti–c-Myb antibody. (F) VE-cadherin+CD31+cells were sorted from differentiating c-mybTet/KO ES cells and recultured on OP9 layers in the absence of Tet. After 3 days, the cultures were stained in situ with antibodies against VE-cadherin (green) and c-Myb (red). VE-cadherin staining was localized to cell-cell junctions and perinuclear regions, whereas c-Myb staining was localized to nucleus. Results shown are representative of at least 3 independent experiments. Original magnification, ×600 (objective, 60×/1.25 NA oil).

Tet-regulated expression of c-Myb protein in c-myb knockout ES cells. (A) Wild-type ES cells (WT), c-myb–/–parental ES cells expressing tTA (Tet/KO), and c-myb–/–ES cells carrying the Tet-regulated c-myb transgene (c-mybTet/KO) were cultured for 2 days in the presence (1 μg/mL) or absence of Tet. Cells were lysed and subjected to immunoprecipitation and Western blotting with an anti–c-Myb antibody. Two isoforms of the c-Myb protein, p75 (filled arrow) and p89 (open arrow), were detected in wild-type cells, whereas only p75 was detected in c-mybTet/KO cells cultured in the absence of Tet. Total-cell lysates were blotted with antiactin to normalize the initial amount of protein. (B) c-mybTet/KO ES cells were cultured for 2 days in the presence of indicated concentrations of Tet (0-1000 ng/mL). Cells were lysed and subjected to immunoprecipitation and Western blotting as described under “Materials and methods.” (C) c-mybTet/KO ES cells were cultured in the presence (top panels) or absence (bottom panels) of Tet (1 μg/mL) for 2 days. The culture conditions were then shifted to either with Tet (1 μg/mL) (bottom panels) or without Tet (top panels), and incubation continued for another 6 to 24 hours. Cells were lysed and subjected to immunoprecipitation and Western blotting. (D) c-mybTet/KO ES cells were cultured in the presence (top panels) or absence (bottom panels) of Tet (1 μg/mL) for 2 days. The culture conditions were then shifted to either with Tet (1 μg/mL) (bottom panels) or without Tet (top panels), and incubation continued for another 12 to 96 hours. Cells were then analyzed for EGFP expression by flow cytometry. Histograms show intensity of EGFP fluorescence on the cells harvested at indicated time points (green) together with cells that were kept in the presence of Tet as a negative control (black). (E) c-mybTet/KO ES cells were allowed to differentiate on OP9 stromal cell layers in the presence of Tet (1 μg/mL) for 4 days. Flk-1+cells were sorted by FACS and recultured with OP9 cells in the presence (top panel) or absence (bottom panel) of Tet (1 μg/mL). After 24 hours, the culture conditions were shifted to either with Tet (1 μg/mL) (bottom panel) or without Tet (top panel), and incubation continued for another 6 to 24 hours. Cells were then lysed and subjected to immunoprecipitation and Western blotting with an anti–c-Myb antibody. (F) VE-cadherin+CD31+cells were sorted from differentiating c-mybTet/KO ES cells and recultured on OP9 layers in the absence of Tet. After 3 days, the cultures were stained in situ with antibodies against VE-cadherin (green) and c-Myb (red). VE-cadherin staining was localized to cell-cell junctions and perinuclear regions, whereas c-Myb staining was localized to nucleus. Results shown are representative of at least 3 independent experiments. Original magnification, ×600 (objective, 60×/1.25 NA oil).

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