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Fig. 4 hSef-b reduces cyclin D1 levels and inhibits the Ras/MAPK pathway in FGF2-stimulated cells. Control and hSef-b inducible NIH 3T3 cells were serum starved for 24 h in the presence and absence of tet and then stimulated with FGF2 (20 ng/ml) for the indicated time periods. (A) The effect of hSef-b on the levels of cyclin D1 protein. The levels of cyclin D1 were evaluated in total cell lysates over 20 h of stimulation. Cyclin D1 and cdk proteins were analyzed by immunoblotting with anti-D1 monoclonal antibody or rabbit anti-cdk4. (B) The effect of hSef-b on FGF2-induced signaling pathways. Equal amounts of total cell lysates were analyzed by immunoblotting. The membranes were successively incubated with the indicated antibodies. (C) Activation of ERK1/2 MAPK in the control cultures. In cells transfected with control vector, there was no effect of tet removal on ERK1/2 activation. Each experiment was repeated at least twice and by using two independent clones of hSef-b inducible cells. P-ERK1/2, P-Akt, P-p38, and P-MEK1/2 are antibodies directed against the phosphorylated (P) form of the kinases.