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Fig. 2. Proteasomal activity in subcellular fractions. HEp-2 cells were grown to subconfluence, followed by biochemical purification of cytoplasmic (A), nucleoplasmic (B), nucleolar (C) and nuclear envelope (D) cell fractions as detailed in Materials and Methods. The purity of cell fractions was monitored by immunoblotting. Proteins from preparations of cellular fractions were separated by SDS-PAGE electrophoresis, transferred to nitrocellulose and immunolabelled with signature proteins of subcellular compartments, such as ß-tubulin (cytoplasm), SC35 (splicing speckles, nucleoplasm), fibrillarin (nucleolus) and lamin A/C (nuclear envelope). Proteasomal activity of subcellular fractions was determined by their incubation with fluorogenic precursor Suc-LLVY-AMC at 37°C and fluorometric detection of cleaved substrates. The resulting product curves (filled circles) were observed over time. Fluorescence intensity values represent mean values of three independent experiments with standard errors between 0.00 and 1.25 (fluorescence intensity, arbitrary units). To control substrate specificity proteasome-inhibitor lactacystin (10 µM) was added after 2 hours (open arrowheads) to double determination assays (open circles). Biochemically purified 20S proteasomes were included as positive controls (grey curves). 20, 30, 40, 50, 60, 70 molecular mass in kDa.
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