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. 1999 Nov 15;147(4):775–790. doi: 10.1083/jcb.147.4.775

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© 1999 The Rockefeller University Press

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/).

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graphic file with name JCB9907019.f3a.jpg — (A) Subcellular fractionation of flt3-induced DC from CatS^−/− and wt mice. PNS of flt3-induced DC from CatS^−/− and wt mice were fractionated using a 27% Percoll gradient as described, yielding the high-density peak A (left panel, fractions 1 and 2). The low-density peak of β-hexoaminidase activity (fractions 9 and 10) was applied subsequently onto a 10% Percoll gradient (right panel) to generate peaks B (fractions 1 and 2 of the 10% gradient) and C (fractions 11 and 12 of the 10% gradient). The β-hexosaminidase activity (beta-hex) and the distribution of incorporated radioactivity after metabolic labeling of the individual fractions are expressed as percentage of the total amounts retrieved from each gradient. (B) Comparison of the subcellular fractionation profiles from DC of different origin. DC from wt mice were either isolated from spleens after treatment with flt3 ligand (flt3 DC) or generated from bone marrow precursors of nontreated animals (BM DC). Subcellular fractions were prepared and assayed for β-hexosaminidase activity and incorporated radioactivity as in Fig. 3 A. (C) Characterization of subcellular fractions. Subcellular fractions were generated from flt3-induced splenic DC of wt mice as described. The distribution of lamp-1, M6PR, and TfR was assessed by Western blot followed by densitometry.

graphic file with name JCB9907019.f3b.jpg — (A) Subcellular fractionation of flt3-induced DC from CatS^−/− and wt mice. PNS of flt3-induced DC from CatS^−/− and wt mice were fractionated using a 27% Percoll gradient as described, yielding the high-density peak A (left panel, fractions 1 and 2). The low-density peak of β-hexoaminidase activity (fractions 9 and 10) was applied subsequently onto a 10% Percoll gradient (right panel) to generate peaks B (fractions 1 and 2 of the 10% gradient) and C (fractions 11 and 12 of the 10% gradient). The β-hexosaminidase activity (beta-hex) and the distribution of incorporated radioactivity after metabolic labeling of the individual fractions are expressed as percentage of the total amounts retrieved from each gradient. (B) Comparison of the subcellular fractionation profiles from DC of different origin. DC from wt mice were either isolated from spleens after treatment with flt3 ligand (flt3 DC) or generated from bone marrow precursors of nontreated animals (BM DC). Subcellular fractions were prepared and assayed for β-hexosaminidase activity and incorporated radioactivity as in Fig. 3 A. (C) Characterization of subcellular fractions. Subcellular fractions were generated from flt3-induced splenic DC of wt mice as described. The distribution of lamp-1, M6PR, and TfR was assessed by Western blot followed by densitometry.