Fig. 5.

CDK activity is required for IFN-β mRNA translation. (A) In vitro translation was performed for a control luciferase mRNA in rabbit reticulocyte lysates pretreated with DMSO, R547 (10 nM), or cycloheximide (CHX; 10 µg/mL). Reactions were assayed for luciferase activity 30 min later. (B) THP-1 cells were depleted of endogenous amino acids by incubation with Met/Cys-free media in the presence of DMSO, R547, or CHX for 2 h. ³⁵S-labeled amino acids were added to culture media, allowed to incorporate into newly synthesized proteins for 30 min, and cell lysates were analyzed by SDS/PAGE. Total protein levels were visualized by Coomassie staining (Left), ³⁵S incorporation was determined by autoradiography (Right). (C) THP-1 cells were transfected with DNA (4 µg/mL) in the presence of DMSO or R547. Cytoplasmic extracts were prepared 2 h after transfection, and polysome profiling was performed by sucrose gradient centrifugation. (D) qRT-PCR for the indicated mRNA targets were performed on each fraction. Values are represented as the ratio of mRNAs associated with polysomes over total mRNA for that message. Data are representative of at least two independent experiments.