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Growth-factor deprived mCFU-E cells (5x106 cells per condition) and BaF3-EpoR cells (1x107 cells per condition) were stimulated with different Epo doses and absolute concentrations were determined for pEpoR (B), pAKT (C), ppERK (D). The scale for pS6 (E) was estimated in arbitrary units. GTP-Ras (F) and ppERK were determined upon stimulation with indicated, color-coded Epo doses. pEpoR was analyzed by immunoprecipitation followed by immunoblotting, GTP-Ras was analyzed after pulldown using a
Transcriptional response to a sudden increase in extracellular ligand (hormone), for the six network designs of (A). The transcriptional response is taken to equal the ratio ReNrL/Retotal, i.e., the fraction of REs attaching ligand-bound NR. The ligand concentration was increased from 0 to 0.005 nM and maintained constant at the latter level. The observation that design 6 is higher than all other designs at long times is robust for parameter changes up to a factor of 3.
After the removal of the extracellular antibiotic, efflux and inhibition dynamics combine to delay the synthesis of ribosomes in a concentration-dependent manner (panel ii). Colors indicate increasing antibiotic concentration, as shown in panel ii.
For all experiments, primary CFU-E cells were starved and stimulated with 5 U/ml Epo. At the indicated time points, samples were subjected to quantitative immunoblotting. Experimental data (black circles) with estimated standard errors and trajectories of the best fit (solid lines) are represented. Mass spectrometry data represent replicates of four independent experiments.
3 chemostat experiments:
each in 4 biological replicates incl. 1 fed with labelled glucose
T = 37°C
pH = 7.1
V_R = 300 mL (dasgip parallel bioreactor system)
V_G = 9 sL/h (0.5 vvm)
M9 Minimal medium + 3,4-dihydroxybenzoate (chelating agent) + 1g/L Glucose
n = 1000 rpm
"reference" without additional sodium chloride as control
"stress" supplemented with 1.2M sodium chloride
"osmoprotection" supplemented with 1.2M sodium chloride and 1mM glycine betaine
Person responsible: Michael Kohlstedt
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