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General Information info

Manuscript title Division of labor by dual feedback regulators controls JAK2/STAT5 signaling over broad ligand range.
PubMed ID 21772264
Journal Molecular Systems Biology
Year 2011
Authors Julie Bachmann, Andreas Raue, Marcel Schilling, Martin E. Boehm, Clemens Kreutz, Daniel Kaschek, Hauke Busch, Norbert Gretz, Wolf D. Lehmann, Jens Timmer and Ursula Klingmueller
Affiliations Division of Systems Biology of Signal Transduction, DKFZ-ZMBH Alliance, German Cancer Research Center, Heidelberg, Germany
Keywords apoptosis, erythropoietin, mathematical modeling, negative feedback, SOCS, simulation and data analysis, signal transduction
Full text article Downloadarticle Bachmann_2011.pdf
Project name not specified

Experiment Description info

Organism Mus musculus
Strain E13.5 Balb/c
Data type enzyme/protein concentrations
Data units (a.u.)
Execution date not specified

Experimental Details info

Temperature (0C) 37.0
pH not specified
Carbon source not specified
Culture mode batch
Process condition not specified
Dilution rate (h-1)
Working volume (L) not specified
Biomass concentration (g/L) 4.0×10^7 (cells/L)
Medium composition

Cultivated for 12–14 h in IMDM (Invitrogen), 30% fetal calf serum (FCS), and 50 μM β-mercaptoethanol supplemented with 0.5 U/ml Epo (Cilag-Jansen).

General protocol information Measurement method: immunoblotting

Methods description - Notes

Time course experiments, cell lysis and quantitative immunoblotting - erythroid progenitor cells were washed three times in medium and starved in Panserin 401 (Pan Biotech) supplemented with 50 µM ß-mercaptoethanol and 1 mg/ml BSA (Sigma-Aldrich) for 1 h. Subsequently, cells (4x10^7/ml) were stimulated with 5 U/ml Epo (Janssen-Cilag) at 37ºC and for each time point 0.5x10^7 CFU-E cells were lysed in 2 x lysis buffer (2% NP40, 300mM NaCl, 40mM Tris–HCl pH 7.4, 20mM NaF, 1mM EDTA pH 8.0, 2mM ZnCl2 pH 4.0, 1mM MgCl2, 2mM Na3VO4, 20% glycerol, 2 µg/ml aprotinin and 200 µg/ml AEBSF). Immunoprecipitation was performed by adding the respective antibody, Protein A or Protein G sepharose (GE Healthcare) and the corresponding calibrator protein GST-EpoR, SBP-JAK2, GST-STAT5b, SBP-CIS or SBP-SOCS3 to the lysates. Antibodies used were anti-EpoR M-20 (Santa Cruz), anti-JAK2 (Upstate/Millipore), anti-STAT5 C-17 (Santa Cruz), anti-SHP-1 C-19 (Santa Cruz), anti-CIS serum [1], anti-SOCS3 (Zymed) and for immunoblotting anti-pTyr 4G10 (Upstate/Millipore), anti-CIS N-19 (Santa Cruz) and anti-SOCS3 (Abcam) as well as secondary horseradish peroxidase coupled antibodies (anti-rabbit HRP, anti-mouse HRP, anti-goat HRP, protein A HRP; Amersham Biosciences). Sample loading on SDS–PAGE was randomized as described elsewhere [2]. Blots were developed using ECL Western Blotting Detection Reagents (GE Healthcare) and subsequently detected on a Lumi-Imager F1TM (Roche Diagnostics). Quantification was performed using the LumiAnalyst 3.1 software (Roche Diagnostics). Antibodies were removed by treating the blots with ß-mercaptoethanol and SDS as described previously [3].

---------------------References------------------
[1] Ketteler R, Moghraby CS, Hsiao JG, Sandra O, Lodish HF, Klingmuller U(2003). The cytokine-inducible Scr homology domain-containing protein negatively regulates signaling by promoting apoptosis in erythroid progenitor cells. J Biol Chem 278: 2654–2660. http://doi.org/b6fp9v
[2] Schilling M, Maiwald T, Bohl S, Kollmann M, Kreutz C, Timmer J, Klingmuller U (2005) Quantitative data generation for systems biology: the impact of randomisation, calibrators and normalisers. Syst Biol (Stevenage) 152: 193–200. http://doi.org/dknn3j
[3] Klingmuller U, Lorenz U, Cantley LC, Neel BG, Lodish HF (1995). Specific recruitment of SH-PTP1 to the erythropoietin receptor causes inactivation of JAK2 and termination of proliferative signals. Cell 80: 729–738. http://dx.doi.org/10.1016/0092-8674(95)90351-8

Data file
Downloadproteomic KIMODATAID81_v0.xlsx
Alternative format(s)
KIMODATAID81_transducted_timeseries.csv
KIMODATAID81_stimulated_timeseries.csv
KIMODATAID81_actinomycinD_timeseries.csv
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Organism



Submission and curation info

Entered by Administrator KiMoSysFirst name: Administrator
Affiliation: INESC-ID/IST
Homepage: http://kdbio.inesc-id.pt/kimosys
Interests: mathematical modeling, accessible data, use of data

Created 2014-04-23 15:16:48 UTC

Updated 2014-06-13 23:32:46 UTC

Version 0

Status (reviewed) 2018-07-07 21:38:44 UTC




Associated Models


Here we can find relevant models associated with Data EntryID 81:

Model
EntryID
Model name Category Model Type Data used for Access Json
31
Authors: Julie Bachmann, Andreas Raue, Marcel Schilling, Martin E. Boehm, Clemens Kreutz, Daniel Kaschek, Hauke Busch, Norbert Gretz, Wolf D. Lehmann, Jens Timmer and Ursula Klingmueller

Original paper: Division of labor by dual feedback regulators controls JAK2/STAT5 signaling over broad ligand range.

transcriptional model of JAK2/STAT5 signaling Signal ordinary differential equations Model building Visto4 {"affiliation":"Division of Systems Biology of Signal Transduction, DKFZ-ZMBH Alliance, German Cancer Research Center, Heidelberg, Germany","article_file_name":null,"authors":"Julie Bachmann, Andreas Raue, Marcel Schilling, Martin E. Boehm, Clemens Kreutz, Daniel Kaschek, Hauke Busch, Norbert Gretz, Wolf D. Lehmann, Jens Timmer and Ursula Klingmueller","biomodels_id":"","category":"Signal","combine_archive_content_type":"application/omex+xml","combine_archive_file_name":"COMBINE_KIMOMODELID31.omex","combine_archive_file_size":1445888,"combine_archive_updated_at":"2014-06-30T17:59:26Z","comments":"","control":null,"dilution_rate":"","id":31,"journal":"Molecular Systems Biology","keywords":"apoptosis, erythropoietin, mathematical modeling, negative feedback, SOCS, simulation and data analysis, signal transduction","main_organism":"Mouse","manuscript_title":"Division of labor by dual feedback regulators controls JAK2/STAT5 signaling over broad ligand range.","model_name":"transcriptional model of JAK2/STAT5 signaling","model_type":"ordinary differential equations","organism_id":null,"project_name":"","pubmed_id":"21772264","review_journal_id":null,"sbml_file_name":"Bachmann_2011.pdf","software":"http://www.matlab.com (MATLAB)","used_for":"---\n- Model building\n","year":2011} Administrator KiMoSys



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