Fig. 1. Cell surface 5T4 oncofoetal antigen is upregulated on ES cells following removal of LIF. (a) Cell surface expression of 5T4 on ES cells: (i) MESC, (ii) D3, (iii) OKO160 and (iv) 129 ES cells were differentiated for 12 days as monolayer cultures by removal of LIF from the growth medium and cell-surface 5T4 was measured using rat anti-m5T4 monoclonal antibody 9A7 (uncoloured population) or control rat IgG (coloured population). Primary antibodies were detected using FITC-conjugated rabbit anti-rat Ig and cell fluorescence measured in a Becton Dickinson FACScan. Viable cells were gated using forward and side scatter and the figure shows the fluorescence of this population. Percentages of each population expressing 5T4 is shown. Day 0, undifferentiated cells; Day 12, 12 days following removal of LIF. (b) Total 5T4 protein expression in ES cells. (i) MESC, (ii) D3, (iii) OKO160 and (iv) 129 ES cells were differentiated for 12 days as monolayer cultures by removal of LIF from the culture medium, lysed (1.2x10⁷ cells/ml) and 20 µl of the lysate separated by unreduced SDS-PAGE. The membrane was probed using rabbit anti-m5T4 polyclonal serum followed by HRP-conjugated sheep anti-rabbit immunoglobulins and developed by enhanced chemiluminescence. Bar charts show the densitometric analysis of the 5T4 bands, with arbitrary density values on the y-axis and days post-removal of LIF on the x-axis. Controls are mouse A9 cells transfected with m5T4 cDNA (positive) or vector control (negative).

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