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Fig. 7. Cholesterol depletion by methyl-ß-cyclodextrin (MCD) and its effect on CNTF-, IL-6- and LIF-signalling. (A,B) MDCK cells, cultured for 72 hours, were plated on 60 mm tissue culture dishes and incubated with mevalonate (0.25 mM) and lovastatin (4 µM) for 48 hours prior to the experiment. Control cells (0 minutes MCD) were not pretreated. Cholesterol was depleted using 10 mM (A) or 20 mM MCD (B) in DMEM containing 4 µM lovastatin and 0.2% BSA at 37°C for different times as indicated. Total cellular lipids were extracted and cholesterol measured enzymatically at 500 nm. The data represent the mean±s.d. of three independent experiments. The percentage decrease in cellular cholesterol levels is also given above each bar. (C) MDCK-gp130 cells were pretreated (+) with lovastatin (Lov) and mevalonate (Mev) as described above or left untreated (—). The cells were stimulated with IL-6 (20 ng/ml)/sIL-6R (500 ng/ml) in DMEM/0.2% BSA for 30 minutes at 37°C (+) or left untreated (—). Nuclear extracts were prepared and equal protein aliquots analysed by SDS-PAGE and western blotting. The membranes were probed with an activation-specific STAT3 antibody and an HRP-conjugated secondary antibody. Proteins were visualised using the ECL+plus system. (D) MDCK-CNTF-R (upper panel), MDCK-gp130 (middle panel) and parental MDCK cells (lower panel) were grown on tissue culture dishes and incubated with mevalonate and lovastatin as described above. Control cells (first two lanes) were left untreated. Lovastatin (4 µM) was also included in all depletion or stimulation solutions except for controls. The cells were cholesterol-depleted using 10-20 mM MCD at 37°C for different times as indicated and afterwards stimulated (+) with CNTF (25 ng/ml), IL-6 (20 ng/ml)/sIL-6R (500 ng/ml), or LIF (50 ng/ml), respectively. Nuclear extracts were prepared and analysed as outlined above.
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