PDI distribution in resting and activated endothelial cells by gradient centrifugation. Organelles in a HUVEC lysate were separated by centrifugation on a Nycodenz density gradient. Percentages refer to percentage per fraction of total activity or antigen recovered in the 14 fractions of the gradient. The numbers 1-14 of the fractions are displayed on the x-axis. (A) Immunoblots of fractions 1-14 (higher to lower density, right to left) using the monoclonal PDI antibody, RL90, in fractions from unactivated (top panel, PDI) or thrombin-activated (second panel, PDI⁺) HUVECs. Middle and bottom panels show immunoblots for distribution of markers for endoplasmic reticulum, SERCA 2b; Weibel-Palade bodies, VWF; or plasma membrane, alkaline phosphatase, with (+) or without thrombin activation of cells. The numbers on the immunoblots refer to the density-gradient fractions. (B) The percentage per fraction of PDI antigen (—) or PDI enzymatic activity (- - -) are shown as the average of 2 experiments. The open circles (○) indicate fractions from unactivated HUVECs, while closed circles (•) show fractions from thrombin-activated HUVECs. The mean density of the fractions is indicated by the gray dashed line. (C). The average percentage per fraction from 2 experiments for markers, alkaline phosphatase (▴), SERCA2b (•), or VWF (■).