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Fig. 3. Overexpression of B-Myb influences endoreplication in megakaryoblastic cells. (A) Diagram of the pIRES-EGFP and B-Myb-IRES-EGFP plasmids. HEL cells were transfected with pIRES-EGFP control plasmid or B-Myb-IRES-EGFP and sorted for GFP expression after 18 hours. CMV Pr, cytomegalovirus promoter. (B) 50 µg of total protein from GFP+ and GFP– populations were subjected to SDS-PAGE and detected by western blot with antibodies against B-Myb and ß-actin. (C) Cell-cycle distribution of sorted GFP– cells and GFP+ cells treated with TPA for 72 hours; the middle and right panels correspond to GFP+ cells from transfections with the control vector and B-Myb-IRES-EGFP, respectively. The vertical axis indicates the relative number of cells and the horizontal axis indicates the relative red fluorescence (FL2) on a logarithmic scale as a measure of DNA content. (D) Flow cytometric analysis of GFP+ cells that were in the presence of TPA for 18 hours, followed by 3 hours in the presence of TPA and BrdU. Incorporation of BrdU was detected by indirect immunofluorescence using a FITC-conjugated mouse anti-BrdU monoclonal antibody and FITC-conjugated mouse IgG1 isotype control (FL1, vertical axis, logarithmic scale), and total DNA content was monitored by propidium iodide staining (FL2, horizontal axis, linear scale). The left and right panels correspond to GFP+ cells from transfections with the control vector and B-Myb-IRES-EGFP, respectively.
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