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. 2012 Jul 31;287(39):32728–32737. doi: 10.1074/jbc.M112.373274

FIGURE 3.

FIGURE 3.

EIF4E-P but not eIF2α-P protein levels are elevated in NPM1+/− cells. A, MNPM1+/− cells express elevated levels of eIF4E-P and c-Myc compared with MNPM1+/+ cells, as measured by Western blot analysis. Equal protein loading was confirmed by probing the blot with β-actin. This experiment has been repeated four times. B, no change in protein levels of eIF2α or P-eIF2α were observed in MNPM+/− cells compared with wild type MNPM1+/+ cells as measured by Western blot analysis. Equal protein loading was confirmed by probing the blot with β-actin. C, quantitative analysis of the relative expression levels of eIF4E and phopsho-eIF4E in four separate experiments compared with β-actin expression using Image J software comparing MNPM1+/+ and MNPM1+/− cells. Expression levels of eIF4E remained unchanged, but those of eIF4E-P increased 1.8-fold in MNPM1+/− cells (p = 0.044). D, C/EBPαp30 up-regulates the eIF4E promoter. Transient co-transfection analysis of eIF4E promoter-reporter and expression plasmids for c-Myc (2 μg) as a positive control, C/EBPαp30 (1 μg), and C/EBPαp42 (1 μg) in HEK293 cells. 48 h post-transfection, reporter gene activity was measured by the dual luciferase assay kit (see “Experimental Procedures”). Normalized firefly luciferase (to that of a co-transfected Renilla luciferase plasmid) values have been represented as a fold change over the enzyme activity of eIF4E promoter reporter plasmid alone (equal to 1). The figure represents the normalized means ± S.E. obtained from an experiment performed in triplicate. This experiment was repeated three times. *, p = 0.009; **, p = 0.016.