Fig. 4.

4E-BP1 enhances cap-independent Cd40 mRNA translation. A, WT and 4E-BP1/2 double knockout (DKO) mouse embryonic fibroblasts were cultured in the presence of TMG or vehicle (Veh) for 24 h. Protein O-GlcNAcylation and expression of eIF4G, tubulin, eIF4E, and 4E-BP1 were assessed by Western blotting of retinal lysates. Protein molecular mass (in kDa) is indicated at left. B, the interaction of 4E-BP1 and eIF4G with eIF4E was examined by immunoprecipitating eIF4E from cell lysates and measuring the amount of eIF4G and 4E-BP1 in the immunoprecipitate (IP) by Western blotting. C, the interaction of 4E-BP1 with eIF4E in B was quantified. D, a hairpin structure was predicted for the Cd40 5′-UTR using the mfold web server. E, a bicistronic reporter was generated wherein a single mRNA encodes for both YFP and CFP. YFP expression is mediated by cap-dependent translation, whereas cap-independent expression of CFP is under the control of the Cd40 5′-UTR. F, WT and 4E-BP1/2 double knockout (4EBP-DKO) mouse embryonic fibroblasts expressing Cd40 reporter were cultured in the presence of TMG or vehicle for 24 h. G, human MIO-M1 Müller cell cultures were cotransfected with the Cd40 reporter, and either a plasmid that expresses an empty vector (EV) or a 4E-BP1 F113A variant that constitutively binds eIF4E. CFP, YFP, tubulin, and 4E-BP1 expression were assessed by Western blotting of cell lysates. The ratio of CFP to YFP expression was quantified from Western blots. The values are means ± S.D. A Student's t test was used to compare differences between groups in C and G. The data in E were analyzed by two-way ANOVA (Table S1). Dunnett's post hoc analysis was used to identify differences between means when significant main and interaction effects were detected. *, p < 0.05 versus vehicle; #, p < 0.05 versus WT.