Fig. 2. B-Myb protein is expressed in primary mature megakaryocytes. (A) E15 foetal liver cells were cultured in vitro for 4 days in the presence of TPO, then immunodepleted and subjected to fractionation on a discontinuous BSA gradient. (B) Propidium iodide staining of cells from Fractions 1 and 4. 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. Peaks representing each ploidy class are labelled. (C) Megakaryocytes from Fractions 1 and 4 were cytospun and stained for acetylcholinesterase. (D) 50 µg of total protein extract from cells in Fractions 1 and 4 were subjected to SDS-PAGE and detected by western blot with antibody against B-Myb. The position of relevant molecular weight standards is shown on the left side of the blot. Coomassie Blue staining was performed with the upper part of the gel as a loading control. (E) Expression of B-Myb was detected in E15 foetal liver (FL) cells cultured in the presence of TPO for 4 days, by indirect immunofluorescence using a B-Myb polyclonal antibody or IgG control and FITC-conjugated goat anti-rabbit IgG (FL1, vertical axis, logarithmic scale), and total DNA content was monitored by propidium iodide staining (FL2, horizontal axis, logarithmic scale). The polygon shows the position of cells stained in parallel with the isotype control. (F) DNA synthesis in mature megakaryocytes. Cells that had differentiated for 5 days were labelled with BrdU for 16 hours. Incorporation of BrdU was detected by indirect immunofluorescence using a mouse anti-BrdU antibody and PE-conjugated goat anti-mouse Ig secondary antibody (red). Nuclei were stained with DAPI (blue). The upper panels are of a mature megakaryocyte that had not been labelled with BrdU. The middle and lower panels are examples of cells that have incorporated BrdU, representing, respectively, a mature polyploidy megakaryocyte and a terminally differentiated, platelet-producing cell.