sE-selectin induces phosphorylation of ERK1/2 and Akt independent of each other.

(A) HMVECs were stimulated with 50 nM sE-selectin for 30 minutes. In the inhibition study, HMVECs were pretreated with either PP2, PD98059, or LY294002 (10 μM) for 2 hours prior to stimulating with sE-selectin. Cell extracts were prepared and the protein content in each sample was quantitated. Of each sample, 20 μg was resolved by 10% SDS-PAGE and probed with rabbit antihuman phospho-ERK1/2 (*p-ERK1/2) or rabbit antihuman phospho-Akt (*p-Akt) antibodies. To verify equal loading, the blots were stripped and reprobed with rabbit antihuman ERK1/2 or Akt antibody. (B) HMVECs transfected with pcDNA3 containing no insert or ERK1/2 or Akt mutant were stimulated with sE-selectin for 30 minutes and cell lysates were prepared. Cell extracts were resolved by 10% SDS-PAGE and probed with rabbit antihuman phospho-ERK1/2 (*p-ERK1/2) or rabbit antihuman phospho-Akt (*p-Akt) antibodies. Equal loading was verified by stripping the blots and reprobing with rabbit anti-ERK1/2 or Akt antibody. (C) HMVECs transfected with Src sense or antisense ODNs were stimulated with sE-selectin for 20 minutes and cell lysates were prepared. Cell extracts were resolved by 10% SDS-PAGE and probed with rabbit antihuman phospho-Src (*p-Src) antibody. Equal loading was verified by stripping the blots and reprobing with rabbit anti-Src antibody. (D) Cell lysates from HMVECs transfected with pcDNA3 containing no insert, Akt, or ERK mutants were resolved by 10% SDS-PAGE and probed with mouse monoclonal anti-HA antibody. Equal loading was verified by stripping the blots and reprobing with mouse monoclonal antitubulin antibody. sE-selectin induced a time-dependent increase in Akt and ERK1/2 phosphorylation via Src kinase.