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. 1997 Dec 15;139(6):1433–1446. doi: 10.1083/jcb.139.6.1433

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Isolation of distinct MHC class II–containing organelles. (a) Mel JuSo cells were allowed to internalize HRP for 5 min, following by a 15-min chase. Cells were homogenized and a postnuclear supernatant was applied to a 0.8–1.2 M sucrose gradient (left top, right bottom). After centrifugation, fractions were analyzed for HRP activity (open bars) and β-hexosaminidase (filled bars). Fractions 1-12 (Pool I) and 13-28 (Pool II) were sedimented at 100,000 g and each pool was applied to organelle electrophoresis. (b and c) Distribution of HRP (b, filled triangles) and β-hexosaminidase activity (c, filled circles) and total amount of protein (b, and c, open diamonds) after electrophoresis of pool I and pool II, respectively. Anode, left, cathode, right. Fractions after electrophoresis were sedimented at 100,000 g and proteins were subjected to 12% SDS-PAGE and transferred to nitrocellulose. The presence of MHC class II molecules (d and e) as well as HLA-DM (f and g) was detected by immunostaining of the membrane.