Stabilin-1 and stabilin-2 in HSECs are necessary for the sequestration of damaged RBCs. (A-B) Verification of liver-specific knockdown using hydrodynamic injection. Liver and spleen were harvested from stabilin shRNAs (shmStab1/2)– or control shRNAs (shCont1/2)–treated mice. Expression of stabilin-1 and stabilin-2 were analyzed by Western blotting (A) and quantitative real-time PCR (B). A representative result is shown. The closed arrowheads indicate stabilin-1 proteins and the open arrowheads indicate stabilin-2 proteins. (C) FITC-labeled damaged RBCs were injected into stabilin shRNAs (shmStab1/2)– or control shRNAs (shCont1/2)–treated mice, after which the livers were harvested 15 minutes later. Frozen sections were stained with Abs against stabilin-1 or stabilin-2 (red) and F4/80 (yellow) and then analyzed by confocal microscopy (top panels). Damaged RBCs are shown in green (bottom panels). Scale bar, 10 μm. (D) Quantitation of stabilin-1 and stabilin-2 protein in the liver of shCont1/2- or shmStab1/2-treated mice (left panel). Immunoblot intensities for Stabilin/Actin were quantitated by densitometry and expressed in arbitrary units. The intensity for shCont1/2-treated controls was set to 100%. A representative result is shown. Microscopic quantitations of damaged RBCs, Kupffer cells, and nuclei in the liver section (right 3 panels). Each experiment was independently performed 3 times. Results represent mean ± SD for 6 mice per group; t test: *P < .01 versus shCont1/2.