Fig. 6. Cell adhesion to epimorphin is mediated by v-integrins. (A) Quantification of HMEC adhesion to recombinant epimorphin in the presence of anti-integrin antibodies. Adhesion to r-Epm is inhibited by antibodies that block v- and β1-integrins (left). In the presence of function-stimulating antibody for β1-integrin (st) most of the cells bound to epimorphin within 2 hours (right). v; clone P3G8, v'; clone AV1. Data are the mean ± s.d., n=6, ^*P<0.05 vs r-Epm only. (B) Epimorphin adhesion-induced FAK phosphorylation is inhibited by antibodies against v- and β1-integrins. (C) Immobilized epimorphin specifically binds to v- and β1-integrins from HMECs lysates. Epimorphin beads selectively pull down 120-kDa to 140-kDa proteins (non-reduced condition) from HMEC-surface components labeled with membrane-impermeant biotinylation reagents. HRP-labeled streptavidin was used to visualize the proteins (left blot). Immunoblot of the proteins bound to r-Epm beads under the reduced condition. The cell surface proteins that bound to Epm beads appeared to include v- and β1-integrins. FN, fibronectin (right blot). (D) Function-blocking antibodies against v- and β1-integrins block adhesion of HSC-5 and HUVECs, whereas inhibition of v- and vβ5 integrins blocks adhesion of A549 and MCF7 cells. Data are the mean ± s.d., n=6, ^*P<0.05 vs control. (E) The transient silencing of v-integrin results in a dramatic attenuation of epimorphin association of HMECs. The cells infected with lentivirus without (NT) or with the knockdown construct HE4 or HE5 were assessed for the expression of v- and β1-integrins and cellular adhesion to r-Epm at day 3. Data are the mean ± s.d., n=4, ^*P<0.05 vs NT. (F) Epimorphin beads pull down purified vβ5-integrin but not 1β1-integrin, and this integrin-epimorphin association is not affected by addition of RGDS or RGES peptide. v- or 1β1-integrin bound to the beads was detected with antibodies against v-integrin or β1-integrin, respectively.