Cells in culture were starved for 16 h in D-MEM/0.1% FBS and treated as follows: 10 or 40 ng/ml PMA plus 1 μg/ml ionomycin (Iono) for 15-30 minutes; 50 nM of Calphostin C for 30-60 min followed or not by PMA/Iono 30 min; 5 or 10 μM LY294002 for 1 h; 10, 25 or 50 μM PD98059 for 1 h; 1 or 10 μM SB20358 for 30 min; 50 ng/ml KT5720 for 30 min; or DMSO (solvent); and 10 nM IGF-1 (Millipore Co. Billerica, MA, USA) (Luo et al., 2005 (link)) or water (IGF-1 solvent) for 1 h. The amount of DMSO used was never higher than 0.1%. After treatment, cells were washed and used for isolation of nuclear extracts.
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Cellular Signaling Modulation Assays
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Variable analysis
independent variables
- 10 or 40 ng/ml PMA plus 1 μg/ml ionomycin (Iono) for 15-30 minutes
- 50 nM of Calphostin C for 30-60 min followed or not by PMA/Iono 30 min
- 5 or 10 μM LY294002 for 1 h
- 10, 25 or 50 μM PD98059 for 1 h
- 1 or 10 μM SB20358 for 30 min
- 50 ng/ml KT5720 for 30 min
- 10 nM IGF-1 (Millipore Co. Billerica, MA, USA) or water (IGF-1 solvent) for 1 h
- Amount of nuclear extracts
- Cells were starved for 16 h in D-MEM/0.1% FBS
- The amount of DMSO used was never higher than 0.1%
- DMSO (solvent)
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