Figure S1. Specificity of polyclonal anti stabilin-1 and anti stabilin-2 antibodies Lysates of 293T cells transfected with empty vector, mouse

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1 Figure S1. Specificity of polyclonal anti stabilin-1 and anti stabilin-2 antibodies Lysates of 293T cells transfected with empty vector, mouse stabilin-1, or mouse stabilin-2 were immunoblotted using anti stabilin-1 (1b-R1) or anti stabilin-2 (16-R2) antibody. Each antibody recognized a single band and did not show cross-reactivity with mouse stabililin-1 or stabilin-2.

2 Figure S2. Measurement of PS exposure on the cell surface of damaged RBCs Mouse (A) and human (B) RBCs were incubated at 37 C for 4 or 5 days to induce exposure of PS on the cell surface. Measurement of PS exposure on the cell surface was performed using annexin-fitc by flow cytometry.

3 Figure S3. Kupffer cells engulf damaged RBCs and PS-coated beads in the liver Control (PBS-treated) mice were injected with FITC-labeled damaged RBCs (A) or PS-coated beads (B), after which livers were harvested 15 minutes later. Frozen sections were stained with antibodies directed against F4/80 (yellow) (a marker for Kupffer cells) and analyzed by confocal microscopy. X-Y and Y-Z sections clearly showed that damaged RBCs or PS-coated beads were engulfed by Kupffer cells. drbc, damaged RBC.

4 Figure S4. Spleen uptake of phospholipid-coated beads is independent of PS Mice were injected with NBD-PC labeled PS- or PC-beads (green), after which the spleens were harvested 15 minutes later. Frozen sections were stained with anti stabilin-2 antibody. PC- and PS-beads and Stab2 + cells (red) were visualized by confocal microscopy. Spleen effectively sequestered PC-beads or PS-beads, indicating that uptake in the spleen was independent of PS. Scale bar, 20 µm.

5 Figure S5. Expression of stabilin-1 and stabilin-2 in HSECs and P388D1 cells HSECs (A) and P388D1 (B) cells were grown in an 8-well chamber slide. HSECs were stained with anti-human stabilin-1 (N-13) or anti-human stabilin-2 (5G3) antibody. P388D1 cells were stained using anti-mouse stabilin-1 (1b-R1) and anti-mouse stabilin-2 (16-R2) antibodies. Each isotype-matched antibody was used as a control (inset panels). HSECs highly expressed stabilin- 1 and stabilin-2 (A). Although it was previously reported that P388D1 cells express stabilin-2 at the mrna level (1), stabilin-1 and stabilin-2 proteins were not observed in P388D1 cells (B). Scale bar, 10 µm.

6 Figure S6. Confirmation of shrna-induced suppression of mouse stabilin-1 and stabilin-2 expression (A) Schematic drawing of shrna for knockdown of mouse stabilin-1 (shmstab1) and its scrambled control shrna (shcont1). (B) Schematic drawing of shrna for knockdown of mouse stabilin-2 (shmstab2) and its scrambled control shrna (shcont2). (C) Confirmation of shrna-induced suppression of mouse stabilin-1 expression. 239T cells were transfected with psuper/shmstab1 (shmstab1) or psuper/shcont (shcont1), along with expression vector of mouse stabilin-1. The expression of mouse stabilin-1 was evaluated by immunoblotting with anti-mouse stabilin-1 antibody (1b-R1). (D) Confirmation of shrna-induced suppression of mouse stabilin-2 expression. 239T cells were transfected with psuper/shmstab2 (shmstab2) or psuper/shcont2 (shcont2), along with expression vector of mouse stabilin-2. The expression of mouse stabilin-2 was evaluated by immunoblotting with anti-mouse stabilin-2 antibody (16-R2).

7 Figure S7. Blood decay of normal and damaged RBCs FITC-labeled normal or damaged RBCs were injected into mice. At the indicated times, 5 µl of venous blood was sampled from the tail vein and analyzed by flow cytometry for the fraction of fluorescent RBCs. Each experiment was independently performed three times. Data were normalized to the level at 1 hour after injection and represent mean ± s.d. for six mice per group.

8 Figure S8. Sequestration of damaged RBCs in the spleen of shmstab1/2-treated and shcont1/2-treated mice FITC-labeled damaged RBCs were injected into stabilin shrnas (shmstab1/2) or control shrnas (shcont1/2)-treated mice. Spleens were harvested 15 minutes later. Frozen sections were stained with antibodies against stabilin-1 or stabilin-2 (red) and analyzed by confocal microscopy. Damaged RBCs are shown in green. Scale bar, 50 µm.