rTMD1 inhibits EGF-mediated signaling in HUVECs. (A) EGFR was modified by LeY. HUVECs lysate was immunoprecipitated with Abs against EGFR, ICAM2, or IgG, and then immunoblotted with LeY Ab. ICAM2 acts as a positive control and IgG as a negative control. (B-C) HUVECs were preincubated with various concentrations of Pichia-expressed rTMD1 for 30 minutes, and then stimulated with 10 ng/mL EGF for 10 minutes. (B) rTMD1 concentration-dependently inhibited phosphorylation of tyrosine 1173 on EGFR. EGFR in HUVECs lysate was immunoprecipitated with EGFR Ab, and then immunoblotted with an Ab against the phosphor-tyrosine1173 on EGFR. (C) rTMD1 decreased activation of Akt induced by EGF. The similar results were obtained in at least 3 different experiments, and the relative density of each band was quantified and normalized. The densitometry values are mean ± SD. ***P < .001 compared with the control; ##P < .01 and ###P < .001 compared with EGF alone.