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).

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