Differentiated Dictyostelium cells were incubated in DB with 2 mM caffeine for 20 min at 22°C, washed twice with ice-cold DB, and resuspended at 5×107 cells/ml. Cells were shaken at 200 rpm at 22°C and stimulated with 1 μM cAMP 39 (link). Aliquots were taken at different time points and lysed in SDS-PAGE sample buffer. Proteins were analyzed by immunoblotting with antibodies to PKBA and PKBR1. As extensively described in many studies 23 (link), phosphorylation of PKBA and PKBR1 were detected using anti-phospho-RPS6KB1 antibodies (Cell Signaling, 9205) and anti-phospho-PKC antibodies (Cell Signaling, 2060) because of the similar amino acid sequences around the phosphorylation sites (Fig. S4 ).
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Caffeine Modulates PKB Phosphorylation
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Amino acid sequences
Anti antibodies
Antibodies
Buffer
Caffeine
Cells
Cold
Dictyostelium
Phosphorylation
Proteins
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Variable analysis
independent variables
- Incubation of Dictyostelium cells in DB with 2 mM caffeine for 20 min at 22°C
- Stimulation of cells with 1 μM cAMP
- Phosphorylation of PKBA and PKBR1 detected using anti-phospho-RPS6KB1 and anti-phospho-PKC antibodies
- Shaking of cells at 200 rpm at 22°C
- Washing of cells twice with ice-cold DB
- Resuspension of cells at 5×10^7 cells/ml
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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