Figure 5. R15 inhibits RAS signaling and biological transformation.

(A) Effect of CFP-R15 on EGF-stimulated ERK-MAPK activation in HEK293 cells. CFP, CFP-Mbs (NS1 or R15) and MYC-tagged ERK were co-expressed, and phosphorylation of MYC-tagged ERK was detected following MYC IP and western blot with phosphospecific ERK antibodies. CFP and CFP-NS1 were used as controls.

(B) Cells transfected with the indicated oncogene along with CFP or CFP-Mbs were analyzed for ERK activation as in (A). Quantification of results from (B) are presented in Figure S3B. The experiments were repeated three times for each mutant other than KRAS(G12R).

(C) Effects of R15 on ERK-MAPK signaling in isogenic MEFs expressing either a single RAS locus (KRAS or NRAS) or RASless MEFs rescued by expression of oncogenic BRAF(V600E). The experiments were repeated two times for each isogenic MEF cell line.

(D) Effect of R15 on heterodimerization of endogenous CRAF with RAS and BRAF is shown. HEK cells were co-transfected with the indicated expression constructs encoding oncogenic RAS and CFP or CFP-Mbs. After 48 h, cells were serum starved and cell lysates used to immunoprecipitate endogenous CRAF. The CRAF IPs were then examined for presence of HA-tagged RAS (top panel) and endogenous BRAF (middle panel). Levels of pERK were measured in WCLs to demonstrate efficacy of each Mb at inhibiting specific RAS mutants.

(E) NIH/3T3 cells were transfected with the indicated RAS mutants or oncogenic BRAF or MEK along with CFP or CFP-tagged Mb and allowed to sit at confluence for 2 to 3 weeks. Foci were stained with crystal violet and counted.

(F) Quantification of relative foci number from (E). Results represent the ratio of foci number in presence of CFP-Mb versus CFP alone and are the average of three independent biological experiments, each performed in technical triplicate ± SD. p values were determined by a Student’s t test between CFP and CFP-Mb for each oncogene. ***p < 0.001, **p < 0.01, and *p < 0.05.

See also Figure S3.