DataEntryID 81
    General information
      Manuscript title: Division of labor by dual feedback regulators controls JAK2/STAT5 signaling over broad ligand range.
      PubMed ID: http://www.ncbi.nlm.nih.gov/pubmed/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: https://www.kimosys.org/rails/active_storage/blobs/eyJfcmFpbHMiOnsibWVzc2FnZSI6IkJBaHBBcUVFIiwiZXhwIjpudWxsLCJwdXIiOiJibG9iX2lkIn19--087d02f1a52fb38519e520a53604c05336630099/Bachmann_2011.pdf
      Project name: not specified

    Experiment description
      Organism: Mus musculus
      Strain: E13.5 Balb/c
      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: not specified
      Dilution rate (h⁻¹): —
      Working volume: not specified L
      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: 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: http://kimosys.org/repository/81/download?parameter=1184; 
      Alternative formats: http://kimosys.org/repository/81/attached_files/118/download; http://kimosys.org/repository/81/attached_files/117/download; http://kimosys.org/repository/81/attached_files/116/download; 

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      Entered by: Administrator KiMoSys
      Created: 2014-04-23 15:16:48 UTC
      Updated: 2020-04-24 16:10:34 UTC
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      Status: (reviewed) 2018-07-07 21:38:44 UTC
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