Figure 4

HA-treatment induces CD44-moesin interaction. (A) Both U373 and U87 cells were plated on coverslips and treated with HA (100 μg/mL, 24 h) followed by fixation and labeling with moesin and CD44 monoclonal antibodies as described in Materials and Methods. Panel shows images obtained using confocal laser scan microscopy (CLSM) representing localization of moesin (rhodamine, red) and CD44 (FITC, green) in glioma cell lines (a) U373 and (b) U87 in (i) no treatment controls (NTC) and (ii) HA-treated glioma cells. Arrows show membranous co-localization of moesin and CD44 in glioma cells (U373 / U87) (original magnification X600); (B) Co-IP assays for moesin / CD44 was performed using whole cell lysates obtained from HA (100 μg/mL, 48 h) or TNF-α (10 nM, 24 h) treated or no treatment control glioma cells (U373 / U87) using respective antibodies to determine moesin-CD44 interactions. Panel represents immunoblot (IB) of moesin showing a single band (~72 kDa, moesin) in CD44 immunoprecipitates (IP) obtained from HA-treated or TNF-α treated glioma cells (U87 / U373) and whole cell lysates (WCL of U373 cells) used as positive controls. No band of moesin was observed in negative controls, wherein pull-down was carried out using beads only or isotype mouse IgG. Similarly, moesin-immunoprecipitates showed a single band of CD44 in HA treated or TNF-α treated glioma cells (U87 / U373) and whole cell lysates (U373 cells, input used as a positive control).