CD44 facilitates EBA-175-induced signaling to the RBC cytoskeleton. (A) Schematic of 2D-DIGE assays. (B) 2D-DIGE analysis of WT or CD44-null cRBC lysates stimulated with mock or RII EBA-175. Circles represent spots with ≥1.3 fold change in intensity in mock- vs RII EBA-175–stimulated samples in 1 or both genetic backgrounds as identified by DeCyder analysis. (C) Quantification of the fold change in intensity in RII EBA-175–stimulated vs mock-stimulated WT cRBCs, and RII EBA-175–stimulated vs mock-stimulated CD44-null cRBCs. Spots identified with >1.3-fold change in intensity were plotted. (D) 2D-DIGE analysis of protein phosphorylation in WT vs CD44-null cRBCs stimulated with RII EBA-175. The circles indicate 25 spots with ≥2-fold increase in phosphorylation in the stimulated WT cRBCs compared with CD44-null cRBCs. The 6 spots that were sent for identification by mass spectrometry analysis are indicated. (E) High-confidence proteins identified by mass spectrometry of 2D-DIGE spots with differential phosphorylation between EBA-175–stimulated WT vs CD44-null cRBCs. Phospho-volumes were quantified by Applied Biomics using DeCyder software. (F) Western blot for total and phosphorylated ERM complex proteins (merlin, ezrin, radixin, and moesin) WT or CD44-null cRBCs stimulated with mock vs RII EBA-175. Anti-ERM was used at 1:1000, anti–phospho-ERM was used at 1:1000, and anti-GAPDH (1:4000) was used as a loading control, followed by goat anti–rabbit-HRP (1:4000).