Fig. 4. The effects of inhibitors on CD2 levels in the nucleus and cytoplasm. Cos-1 cells were transfected with plasmids encoding a fluorescent control protein (EYFP-Mito, an enhanced yellow fluorescent protein fused with a mitochondrial targeting sequence from subunit VIII of cytochrome c oxidase) with or without rat CD2, grown for 24 hours, and treated with or without 20 µM lactacystin for 15 minutes. After fixation, CD2 was indirectly immunolabelled with Cy3 (red), and an equatorial (confocal) section through nuclei collected. Only Cy3 fluorescence is shown. In some cases, 100 µM DRB or 5 µg/ml actinomycin D (act D) was added 15 minutes before lactacystin. (A) Cell transfected with plasmids encoding only the control protein (lines mark cell and nuclear peripheries). It contains intensely fluorescing mitochondria (not visible), and no CD2 (average nuclear and cytoplasmic intensities of 0.009 and 0.04 arbitrary units/pixel respectively). (B) Cell transfected with plasmids encoding CD2 (appears white) and the control protein (yellow, not visible); most CD2 is in the ER, however, some faint fluorescence is found in nuclei, and quantitative analysis shows this to have 9x the intensity of that seen in an equivalent area in A. 24% (untransfected) cells in this population expressed no EYFP-Mito or CD2 (i.e., with CD2 labelling like that in A); this population was not analysed further. Scale bar: 10 µm. (C) Intensities in individual cells. Each panel contains results from >75 cells transfected with CD2, each point indicates intensity (arbitrary units/pixel) in nuclear and cytoplasmic areas of one cell, and each arrowhead the average intensity.