Chemical cross-linking of P-selectin in platelets, endothelial cells, HEL cells, and heterologous cells expressing P-selectin.

Purified resting platelets in suspension and nonactivated HUVECs in monolayer (A) and RIN5F cells transfected with P-selectin, HEL cells, and CHO cells expressing P-selectin (C) were treated with the sulfhydryl-specific, membrane-permeable cross-linker DPDPB. The cells were then lysed in buffer containing 1% Triton X-100 in the presence of alkylating agents. Lysates were separated by SDS-PAGE on 6% gels, followed by immunoblotting using P-selectin monoclonal antibodies AK4 for platelets and AC1.2 for all other cells. (B) Lysates from cross-linked, resting platelets were treated with DNase I, separated electrophoretically, and immunoblotted as described in (A).