Figure 3.

S55746-CalPegA combination inhibits global protein synthesis and increases eIF4E/4EPB1 interaction. (A) MOLM14 and MonoMac6 cells were treated with S63845, S55746, and CalPegA alone or in combination (IC₅₀ values) for 24 h followed by a 20 min incubation with puromycin (1 µg/mL). Cell lysates were subjected to immunoblotting with the anti-puromycin antibody (SUnSET [surface sensing of translation] assay). Actin was used as a loading control. The bar diagram shows the mean ± SEM for at least three independent experiments, and unpaired t-tests were used to compare puromycin intensity in the vehicle group to the treatment groups. ** p < 0.01, * p < 0.05. (B) Protein lysates (250 µg) from MOLM14 and MonoMac6 cells treated for 24 h, as in (A), were incubated with antibody against 4EBP1 overnight followed by incubation with Protein A beads. The beads were washed, resuspended in SDS sample loading buffer, and boiled, and then the proteins were separated on 4–15% polyacrylamide gels. Membranes were immunoblotted with antibodies against eIF4E and 4EBP1, and results are expressed as the ratio of eIF4E to 4EBP1. The bar diagram represents densitometric quantification of three independent experiments. *** p < 0.001, ** p < 0.01, * p < 0.05.