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.

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