FIGURE 4.

LMW-HA stimulates CD44 and Src-dependent phosphorylation of EphA2. Panel A, HPMVEC were treated with 100 nm LMW-HA or 1 μg/ml Ephrin-A1-Fc for 5, 15, or 30 min in the presence or absence of the Src inhibitor, PP2 (1 μm). Cells lysates were then prepared and immunoblotted (IB) with phospho-EphA2 (Tyr⁵⁹⁴), phosphor-EphA2 (Ser⁸⁹⁷), EphA2, Phospho-Src (Tyr⁴¹⁸), Src, or actin antibodies. LMW-HA and Ephrin-A1 promote Src-dependent tyrosine phosphorylation of EphA2. Panel B, HPMVEC were pretreated with either control siRNA or CD44 siRNA and then treated with LMW-HA (100 nm) or Ephrin-A1-Fc (1 μg/ml) for 15 min. Cell lysates were prepared in 1% Nonidet P-40 lysis buffer and incubated with anti-EphA2-cross-linked beads for immunoprecipitation. The resulting immune precipitations (IP) were then blotted with phospho-EphA2 (Tyr⁵⁹⁴), CD44v10, Src, or EphA2 antibodies. CD44 siRNA inhibits LMW-HA and Ephrin-A1-Fc-mediated Src recruitment to EphA2. Panel C, graphical depiction of quantity of protein normalized to EphA2 staining intensity (arbitrary units) from experiments described in panel B performed in triplicate and quantitated using computer-assisted densitometry. The asterisks (*) indicate a statistically significant difference (p < 0.05) from control.