Figure 3

E-selectin mediates Src kinase-dependent phosphorylation of Akt through DR3. A) HT29 cells were left untreated or treated for different periods of time (5, 10,15 and 20 min) with rhE-selectin/Fc (5 μg/ml). The proteins were extracted and separated by SDS-PAGE and transferred onto a nitrocellulose membrane. Akt activation was analysed by western blotting with specific anti-phospho (Ser 473)-Akt antibody. The blots was then stripped and reprobed with antibody recognizing total Akt as loading control. B) HT29 cells were pre-incubated or not for 30 minutes with anti-DR3 (hDR3) or anti-DR3 (DR3ecd) before being incubated with rhE-selectin/Fc (5 μg/ml for 15 min). The proteins were extracted and processed as in A. C) HT29 cells were pre-treated for 2 hours with DMSO (0.1%) or LY249002 (20 μM) before being incubated with rhE-selectin/Fc (5 μg/ml for 15 min). The proteins were extracted and processed as in A. D) HT29 cells were pretreated for 2 hours with DMSO (0.08%) or PP2 (10 μM) to inhibit Src family kinases. Thereafter, the cells were left untreated or treated with E-selectin (5 μ/ml for 15 min). The proteins were extracted and processed as in A.