Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Depletion of cellular cholesterol and lipid rafts increases shedding of CD30.

doi: 10.4049/jimmunol.172.7.4324

Figure Lengend Snippet: FIGURE 6. Localization of CD30 in membrane rafts. A–C, Membrane rafts of 3 107 L428 cells were isolated by applying cell lysates to a sucrose gradient and immunoblotted under nonreducing conditions. The indicated molecular mass markers were internal sCD30 standard (90 kDa) for determination of CD30 and commercially available TACE ectodomain standard (75 kDa) for determination of TACE. A, Proteins of fractions 3–11 were separated by nonreducing SDS-PAGE and immunoblotted with a mixture of HRP-labeled anti-CD30 Ki-2 and Ki-4 mAb. B, Proteins of fractions 3–5 (raft fractions) and 9–11 (nonraft fractions) were separated by nonreducing SDS-PAGE and immunoblotted with HRP-labeled anti- TACE MAB9302 mAb. As a control, the TACE-ectodomain standard was used. C, Two microliters of each fraction of L428 cells were dot blotted and stained with peroxidase-labeled cholera toxin subunit B as a positive control for lipid rafts. In fractions 3–5, rafts were isolated. D, L428 cells (1.5 107 per milliliter) were incubated in prewarmed RPMI 1640 con- taining 0.1% BSA in the absence or presence of MCD (10 mM) or PMA (5 ng/ml) at 37°C for 5 or 60 min as indicated. Membrane rafts of 3 107

Article Snippet: The anti-TACE mAb MAB9302, the recombinant human TACE ectodomain Western blotting standard, and tissue inhibitor of metalloproteinases (TIMP)-1 and TIMP-3 were purchased from R&D Systems (WiesbadenNordenstadt, Germany).

Techniques: Membrane, Isolation, SDS Page, Labeling, Control, Staining, Positive Control, Incubation