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. Author manuscript; available in PMC: 2008 Aug 1.
Published in final edited form as: Kidney Int. 2007 Mar 28;72(2):157–165. doi: 10.1038/sj.ki.5002229

Figure 6. Ras and Raf activity in cystic cells stimulated with IGF-1 and cAMP.

Figure 6

(A) Ras activation by IGF-1 and cAMP was investigated using a commercial Ras activity assay which detects GTP-Ras. Total Ras was detected using a pan-Ras antibody. One representative experiment of three is shown. As a positive control, cell lysate was treated with GTP-γ-S prior to pull down with GST-Raf1-RBD (lane ‘GTP’). The numbers displayed show the mean increase in GTP-Ras (corrected to total Ras) in three separate experiments as assessed by densitometry. The signal from control unstimulated cells was corrected to a value of 1. IGF-1 (50 ng/ml) stimulated Ras activity by two fold in cystic (SKI-001) but not in normal cells. DbcAMP (1mM) did not activate Ras in either cell line. (B) The Raf inhibitor, BAY 43-9006, inhibited IGF-1 and dbcAMP stimulated proliferation in cystic cells. Data shown is from three experiments performed in triplicate. (C) Preincubation with BAY 43-9006 (0.5 μM for 2h) abolished IGF-1 stimulated p44/42 ERK but not pAkt phosphorylation in cystic cells.