3T3-L1 adipocytes were incubated in serum-free media at 37°C with (dashed line) or without (solid line) 100 ng͞ml insulin for 8 h. Isoproterenol (10 M) was then added for the indicated time course at 37°C, and intracellular cAMP was determined by enzyme immunoassay, as described in Materials and Methods. Data are from a typical experiment done in triplicate wells Ϯ SEM and are representative of three separate experiments. (B) 3T3-L1 adipocytes were incubated in serum-free media for 8 h. Where indicated, 100 ng͞ml insulin was added for 15 min before isoproterenol treatment. Cells were then treated with 10 M isoproterenol for 5 min at 37°C, and intracellular cAMP was determined by enzyme immunoassay, as described in Materials and Methods. Data are from a typical experiment done in triplicate wells Ϯ SEM and are representative of three separate experiments.www.pnas.org͞cgi͞doi͞10.1073͞pnas.0630528100 After this, cells were treated with 10 M isoproterenol for 5 min at 37°C. Cells were then washed with PBS at 37°C and restimulated with a second 10 M dose of isoproterenol for an additional 5 min at 37°C; intracellular cAMP was determined by enzyme immunoassay. (B) 3T3-L1 adipocytes were incubated at 37°C in serum-free media for 8 h in the presence (dashed lines) or absence (solid lines) of 100 ng͞ml insulin. Forskolin was added for the final 5 min, and intracellular cAMP levels were determined by enzyme-linked immunoassay. Data are from typical experiments done in triplicate wells and are representative of three separate experiments.
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