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. 2010 Sep 30;5(9):e13050. doi: 10.1371/journal.pone.0013050

Figure 1. E-selectin dependent binding of ESTA-1.

Figure 1

(A) ESTA-1 binding to ES-Endo. To determine the E-selectin dependent ESTA binding, ES-Endo cells were treated with increasing concentration of doxycycline (250-2000 ng/ml) and analyzed for E-selectin expression and ESTA-1 binding. E-selectin overexpressing ES-Endo cells were incubated with Cy3-labeled ESTA-1 (100 nM) for 20 minutes at 37°C. TNF-α (10 ng/ml) induced ES-Endo was used as a positive control. (B) Blocking of ESTA-1 binding by E-selectin antibody. ES-Endo were pre-incubated with 25 µg of E-selectin antibody for 2 hours and incubated with 100 nM of ESTA-1 for 20 minutes. Unbound ESTAs were washed away and slides were prepared for fluorescent imaging to visualize the binding to ES-Endo cells. All images were captured at the same exposure condition for comparison. The final images shown are representative images (at the final magnification: x600) from five random fields of at least three independent experiments. Blue, Hoescht 33342; Red, Cy3-labeled ESTA-1; Green, E-selectin.