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Fig. 7. Effect of ceramide-modulating drugs on PLD of L6 myoblasts. (A) PLD activity was assayed by evaluating phosphatidylbutanol formation in intact cells. The cells were labeled with [3H]palmitate, left untreated (control) or pretreated by 20 µM FB1 or 10 µM C6-ceramide for 3 hours. Butanol (1%) was added, with or without 10-7 M AVP, for the last 30 minutes before lipid extraction and analysis. Alternatively, the cells were pretreated by 10 µM C6-ceramide for 24 hours before labeling. PLD activity is expressed as the percentage of total phospholipid radioactivity present in phosphatidylbutanol. The means ± s.e.m. of three to five independent experiments are shown. **Different from the + AVP value, P
0.001. (B) RT-PCR was performed with total RNA from myoblasts cultured for 3 hours in 1% BSA medium, in the absence (control) or presence of AVP, with or without different agents. Primers specific for either PLD1, PLD2 or β-actin transcripts were used. The graphs show the amounts of PLD1 and PLD2 amplification products normalized by the amounts of β-actin amplification product, as quantified by videodensitometry. The means ± s.e.m. of three experiments are shown for PLD1 (**different from + AVP, P0.01), and the means of two determinations differing by less than 7% are shown for PLD2.
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