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Figure 4 | Cell Communication and Signaling

Figure 4

From: Graded inhibition of oncogenic Ras-signaling by multivalent Ras-binding domains

Figure 4

Adjusted, low-level expressed MSOR maintain their Ras-blocking activity. (A) Tet-off promoter-controlled expression of RBD-monomers and MSOR. COS-7 cells were transfected with pNRTIS 12-derived plasmids encoding E1-R3 and subsequently left untreated or treated with different concentrations of doxycycline (Dox) to modulate MSOR expression levels. (B) The impact of inducible mono- and trivalent wild-type RBD probes on oncogenic Ras-stimulated MAP-kinase signaling. COS-7 cells were transiently transfected with different constructs as indicated. Next, cells were divided into samples that were grown either in the absence (-) or presence (+) of 100 ng/ml Dox. Cells lysates were analyzed by Western blot detecting phosphorylated and total Erk2 and the expression of EGFP-constructs. One representative experiment out of three independent experiments is shown. (C) Influence of inducible mono- and trivalent wild-type RBD probes on oncogenic Ras-stimulated MMP-1 promoter activation. NIH3T3 cells were transiently transfected with the MMP-1-luciferase reporter, the K-RasG12V-encoding construct and Tet-off-controlled EGFP-constructs E1, E1-R1 or E1-R3. Expression of EGFP-constructs was turned on (-Dox) or off (+100 ng/ml Dox) and reporter gene activity was measured. The figure shows the average result from three independent experiments each performed in triplicate. (D) Influence of inducible mono- and trivalent wild-type RBD probes on EGF-stimulated cell invasion. COS-7 cells were transfected with the Tet-off-regulated EGFP-constructs indicated, and cultured in the absence of Dox. Subsequently, EGFP-expressing cells were collected by preparative fluorescence activated cell sorting and cultured in absence or presence of 100 ng/ml Dox to regulate the expression of E1, E1-R1 and E1-R3. Then cells were collected, seeded onto Matrigel-coated Transwells and subjected to invasion in absence or presence of 50 ng/ml EGF. Results represent the means of two entirely independent experiments.

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