Figure 5. 4E-BP1 phosphorylation is modulated by AZD8055/selumetinib, but does not regulate BRAF mutant cell survival.

A, the AZD8055/selumetinib combination cooperatively suppressed 4E-BP1 phosphorylation at T37/46 in the BRAF mutant cell line OCM1A. Cells were treated with the indicated drugs for 24 hours. Western blots were then performed. Total 4E-BP1 was used as a loading control. B, the capability of 4E-BP1 binding to the mRNA cap complex was not impacted by the AZD8055/selumetinib combination in OCM1A cells. A cap binding assay was performed (referenced in the Materials and Methods section). Briefly, cells were treated with the indicated drugs for 24 hours. Cell lysates were created and then incubated with m⁷ GTP sepharose beads to capture all 4E-BP1 and eIF4E proteins that are capable of binding to an mRNA cap complex. The bead-associated proteins were analyzed by Western blot. C, suppression of 4E-BP1 expression did not diminish apoptosis as assessed by PARP cleavage in OCM1A cells. Cells were transfected with siRNA constructs targeting 4E-BP1 or unrelated control constructs for 48 hours. Cells were then treated with the indicated drugs for 24 hours. Western blots were performed.