HeLa cells were serum starved (0.2 % FBS, 16 h) and incubated with forskolin and isobutylmethylxanthine (IBMX, 200 μM, 20 min) followed by EGF (30 min). Reactions were terminated by aspiration of media and addition of 150 μl of ice-cold TCA 7.5% (w/v). cAMP content in TCA extracts was determined by radioimmunoassay (RIA) and normalized to protein [(determined using a dye binding protein assay (Bio-Rad)] exactly as we have done previously (28 (link)). Data is expressed as fmol cAMP/μg total protein.
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Quantifying cAMP Signaling in HeLa Cells
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Corresponding Organization : University of California, San Diego
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Variable analysis
independent variables
- Forskolin
- Isobutylmethylxanthine (IBMX, 200 μM)
- EGF (30 min)
- CAMP content
- Serum starvation (0.2% FBS, 16 h)
- Positive control: Not explicitly mentioned.
- Negative control: Not explicitly mentioned.
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