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Blood, Vol. 112, Issue 8, 3465-3473, October 15, 2008
Ligation of erythrocyte CR1 induces its clustering in complex with scaffolding protein FAP-1
Blood Ghiran et al.
112: 3465
Supplemental materials for: Ghiran et al
Files in this Data Supplement:
- Figure S1. CR1 ligands actively cluster CR1 in circulating erythrocytes (JPG, 159 KB)
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Fresh (A, B, C) or fixed erythrocytes (D, E, F) were incubated with AlexaFluor-488 C3b-beads for 30 min, washed and incubated with AlexaFluor-594 labeled 1F11. Pixel intensity for both, green (C3b beads) and red (CR1) channels across erythrocytes (white line in B and E) was plotted for fresh (A) and fixed (D) erythrocytes. Intensity sum of CR1 clusters (X-axis) and intensity sum of the interacting C3b-beads (Y-axis) were plotted for fresh (C) and fixed (F) erythrocytes. Graphs show representative histograms that were plotted from 5 fresh and 17 fixed erythrocytes.
- Figure S2. Isolation of membrane and cytoskeletal fraction of human erythrocytes (JPG, 35.4 KB)
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(A) White ghosts were lyzed in 0.5% C12E8 detergent and centrifuged at 20.000xg over a 35% sucrose cushion for 100 min at 4°C. The white ghosts (E), the supernatant (M), representing the soluble membrane fraction and the pellet (C), representing the insoluble cytoskeletal fraction were solubilized in loading buffer and separated on a 10% Bis-Tris gel. The proteins were transferred on nitrocellulose membrane and probed for spectrin and CD59, which is GPI-anchored. Spectrin (markers of cytoskeletal fraction) is missing in the membrane fraction, and CD59 is missing in the cytoskeletal fraction (membrane marker).
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