Ruffling membranes in cultured human and rat glial cells

We have previously described how mouse monoclonal antibody Mab S-11E10 specifically stained ruffling membranes and filopodia of cultured human glioma cells. We found that this antibody stains ruffling membranes, not only of human astrocytes and glioma cells, but also of rat astrocytes and rat C6 glioma cells. In addition, we newly prepared rabbit polyclonal antibody for the antigens, which were eluted by Mab S-11E10 affigel 10 (Bio Rad) affinity column chromatography from cultured rat C6 glioma cells. The polyclonal antibody detects 65KDa, 57KDa, 46KDa, 43KDa bands in Western blotting analysis and specifically stained ruffling membranes of human glioma cells on cell spreading. Using the polyclonal antibody, we studied the change of the distribution of the antigens during cell spreading and in fully spread cells. At the beginning of cell spreading, ruffling membranes were specifically stained, but 72 hours after plating the immunofluorescence was localized in membranes of supranuclear regions and small protuberances. Furthermore, we semi-quantitatively measured the contents of the antigens detected by the polyclonal antibody, using dot blotting immunoassay. The contents of the antigens rapidly increased at the beginning of cell spreading and thereafter gradually reduced. The change of the contents seemed to be time--and cell density--dependent and to relate to the extent of cell spreading. These results suggest that the antigens may play an important role in cell spreading of glial cells.