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containing 2% FCS to induce differentiation into myo-
tubes. The medium was changed every other day and
cytidine (0.24 mg/mL medium) was added to the cul-
tures at day 7–9 to suspend cycling cells. The cells were
used in experiments after over night serum starvation at
day 11. They were treated with or without 25 mM com-
pound for 30 min followed by 5 min insulin (25 nM) sti-
mulation. After freezing with liquid N2 the cells were
lysed with a Tris–HCl buffer, pH 7.4, containing 1%
Nonidet-40, 0.25% sodium deoxycholate, 150 mM
NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM sodiu-
morthovanadate, 10 mM b-glycerophosphate, 5 mM
sodium pyrophosphate and complete protease inhibitor
cocktail from Boehringer. The cell extracts were cen-
trifuged at 14,000 g for 10 min and the supernatants
were used in the Delfia assay.
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A lanthanide-based fluorescent assay (Delfia) was used
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body to rabbit IgG (from Cappel) and coated with rab-
bit antibody to IR (sc-711 from Santa Cruz
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streptavidin (from Perkin-Elmer). By addition of
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emission measurement after a delay to avoid back-
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