Erythrocyte FAP-1 levels do not parallel those of CR1. (A) Flow cytometric analysis of CR1 expression on erythrocyte from normal donors with low (LL), intermediate (HL), and high (HH) levels of CR1. Finger prick blood from 3 donors (10 μl from each donor) was stained with anti-CR1 mAb 2B11 and incubated with AlexaFluor-488 goat anti–mouse secondary antibody. Cells were then analyzed by FACS within 30 minutes after completion of the staining procedures. The experiment was repeated 4 times with similar results. (B) Western blot analysis of FAP-1 expression in erythrocyte expressing various levels of CR1. Five microliters of freshly purified erythrocyte from the same donors used for Western blot analysis were mixed with 1× reducing- loading buffer and boiled for 3 minutes. Proteins were separated on a 10% Bis-Tris NuPAGE gel and blotted on nitrocellulose membrane. FAP-1 was detected using a mouse mAb. The experiment was repeated 4 times with blood from various HH, HL, and LL donors and different anti–FAP-1 antibodies. (C) Bar graph representing the densitometry results of the levels of FAP-1 in erythrocyte expressing various levels of CR1 after normalization to actin. The loading control was assessed by quantifying the levels of actin (Quantity One; Bio-Rad). The experiment was repeated 3 times with blood from various HH, HL, and LL donors.