Bertram Klinger, Anja Sieber, Raphaela Fritsche-Guenther, Franziska Witzel, Leanne Berry, Dirk Schumacher, Yibing Yan, Pawel Durek1, Mark Merchant, Reinhold Schaefer, Christine Sers and Nils Bluethgen
Affiliations
Laboratory of Molecular Tumour Pathology, Institute of Pathology, Charité - Universitatsmedizin Berlin, Berlin, Germany, and Institute for Theoretical Biology, Humboldt University Berlin, Berlin, Germany
Keywords
cancer, EGFR signaling, modular response analysis, signal transduction
colorectal cancer cell lines SW480, SW403, HCT116, RKO, LIM1215 and HT29
Data type
enzyme/protein concentrations
Data units
(a.u.)
Execution date
not specified
Experimental Details
Temperature (°C)
37.0
pH
not specified
Carbon source
not specified
Culture mode
batch
Process condition
aerobic
Dilution rate (h⁻¹)
—
Working volume (L)
not specified
Biomass concentration (g/L)
not specified
Medium composition
All cell lines were maintained in DMEM (Dulbecco’s modified Eagle’s medium, Lonza) supplemented with 10% fetal calf serum, 1% ultraglutamine and 1% penicillin/streptomycin and incubated in a humidified atmosphere of 5% CO2 in air at 37 ºC.
General protocol information
Measurement method:
immunoblotting
Methods description - Notes
Immunoblotting - protein extracts of cells were prepared as described for Bioplex analysis. Blotting procedure and materials were as previously described [1,2]. The following primary antibodies were used: rabbit anti-human P-p70S6 (Thr389, Cell Signaling Technology, 1:500) o
... r mouse antihuman GAPDH (Ambion, 1:12 500). Membranes were scanned using Li-COR Odyssey. The signals were quantified using Odyssey software.
-------------------References-------------------
[1] Fritsche-Guenther R, Witzel F, Sieber A, Herr R, Schmidt N, Braun S, Brummer T, Sers C, Bluthgen N (2011). Strong negative feedback from Erk to Raf confers robustness to MAPK signalling. Mol Syst Biol 7: 489. http://doi.org/dvbp78 [2] Stelniec-Klotz I, Legewie S, Tchernitsa O, Witzel F, Klinger B, Sers C, Herzel H, Bluthgen N, Schafer R (2012). Reverse engineering a hierarchical regulatory network downstream of oncogenic KRAS. Mol Syst Biol 8: 601. http://doi.org/f2phtg
Network quantification of EGFR signaling unveils potential for targeted combination therapy.
PubMed ID
23752269
Journal
Molecular Systems Biology
Year
2013
Authors
Bertram Klinger, Anja Sieber, Raphaela Fritsche-Guenther, Franziska Witzel, Leanne Berry, Dirk Schumacher,
Yibing Yan, Pawel Durek1, Mark Merchant, Reinhold Schaefer, Christine Sers and Nils Bluethgen
Affiliations
Laboratory of Molecular Tumour Pathology, Institute of Pathology, Charite´ - Universitatsmedizin Berlin, Berlin, Germany, and Institute for Theoretical Biology, Humboldt University Berlin, Berlin, Germany
Keywords
cancer, EGFR signaling, modular response analysis, signal transduction
Project name
not specified
Experiment Description
Organism
Homo sapiens
Strain
colorectal cancer cell lines SW480, SW403, HCT116, RKO , LIM1215 and HT29
Data type
enzyme/protein concentrations
Data units
(a.u.)
Execution date
not specified
Experimental Details
Temperature (0C)
37
pH
not specified
Carbon source
not specified
Culture mode
batch
Process condition
aerobic
Dilution rate (h-1)
—
Working volume (L)
not specified
Biomass concentration (g/L)
not specified
Medium composition
All cell lines were maintained in DMEM (Dulbecco’s modified Eagle’s medium, Lonza) supplemented with 10% fetal calf serum, 1% ultraglutamine and 1% penicillin/streptomycin and incubated in a humidified atmosphere of 5% CO2 in air at 37 ºC.
General protocol information
Measurement method: immunoblotting
Methods description - Notes
Immunoblotting - protein extracts of cells were prepared as described for Bioplex analysis. Blotting procedure and materials were as previously described [1,2]. The following primary antibodies were used: rabbit anti-human P-p70S6 (Thr389, Cell Signaling Technology, 1:500) or mouse antihuman GAPDH (Ambion, 1:12 500). Membranes were scanned using Li-COR Odyssey. The signals were quantified using Odyssey software.
-------------------References-------------------
[1] Fritsche-Guenther R, Witzel F, Sieber A, Herr R, Schmidt N, Braun S, Brummer T, Sers C, Bluthgen N (2011). Strong negative feedback from Erk to Raf confers robustness to MAPK signalling. Mol Syst Biol 7: 489.
[2] Stelniec-Klotz I, Legewie S, Tchernitsa O, Witzel F, Klinger B, Sers C, Herzel H, Bluthgen N, Schafer R (2012). Reverse engineering a
hierarchical regulatory network downstream of oncogenic KRAS. Mol Syst Biol 8: 601.
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