Aberrant Raf activation suppresses Ras via multiple distinct mechanisms
A: SA-β-gal staining of IMR90 infected with Raf1-CAAX (left panel). BJ fibroblasts, expressing an inducible Raf construct, were treated for 30 or 90 minutes with tamoxifen (4-HT, 1 μM). Ras-GTP levels were assessed using a Ras pull-down (PD) assay. Control immunoblots on total cell lysates (TCL) are shown (middle panel). Ras-GTP levels after 24 hours of 4-HT (right panel).
B: SOS immunoblots of BJ cells expressing the inducible Raf construct in the presence and absence of CIP (added to lysates) (top). p120RasGAP protein levels increase as pAKT levels decrease (bottom).
C: BJ cells expressing the inducible Raf were treated for 42 hours with tamoxifen (1 μM) or ethanol. Real-time PCR was performed in triplicate on the listed genes and fold changes were calculated.
D: Immunoblot analysis of lysates from C.
E: Real-time PCR analysis of genes in response to NF1-loss, as compared to cycling, vector-expressing cells.
F: SA-β-gal staining and immunoblots of BJ cells expressing exogenous Sprouty 2.