A model scheme illustrating the contribution of eIF4G proteins in general and DAP5 protein specifically to the fine balance between cell death and viability in the presence of an apoptotic trigger. Positive and negative feedback loops are marked by plus and minus signs, respectively. Caspase cleavage events are marked by scissors.

A diagram to scale highlighting conserved protein-binding regions of mammalian eIF4G homologs aligned through the eIF3/eIF4A-binding region. Regions binding PABP, eIF4E, and Mnk-1 are marked. (Left and Right) Status of eIF4G protein family in growing and dying cells, respectively. eIF4GI numbering is based on the extended sequence (GenBank accession no. AF104913). Names of the apoptosis-associated eIF4G forms conserving an integral eIF3/eIF4A-binding region that arise after caspase cleavage are indicated below. The disintegrated eIF3/eIF4A-binding region is marked by XXX. Scissors mark caspase cleavage sites based on refs. 7, 9, and 18.

(A) Schematic representation of LS-DAP5 bicistronic transcript. (B) 293 cells were cotransfected with GFP, GFP-DAP5/p97, or GFP-DAP5/p86 and LS-DAP5. Reporter levels and the ratio of translation via DAP5 IRES and cap-dependent translation were determined. The reporter ratio obtained by the GFP vector was set at 100%, and the relative fold increase in the SeAP/luciferase ratio was calculated accordingly (Left). The effects of the GFP-DAP5 forms on each reporter independently, normalized to protein level and transfection efficiency, and relative to the reporter activity obtained by the GFP vector are presented (Middle and Right, respectively). The results represent the average of three independent experiments. Asterisks mark statistically significant results. (C) Total RNA samples of the experimental points were Northern-blotted and reacted with probes raised against SeAP cDNA for detection of the bicistronic transcript and GAPDH cDNA for determination of total amount of loaded RNA. The ratio between the bicistronic and GAPDH signals represents the transfection efficiency. The arrows on the left indicate the positions of the bicistronic and GAPDH transcripts. (D) Protein extract samples of representative experimental points were immunoblotted with anti-DAP5 antibodies. The dashed arrow on the left indicates endogenous DAP5/p97. The full arrow indicates exogenous GFP-DAP5/p97 and GFP-DAP5/p86.

(A) Schematic representation of LL bicistronic transcripts (Left). 293 cells were cotransfected with GFP and each LL-based bicistronic vector. Reporter levels were assessed, and the firefly/Renilla luciferase ratio was calculated. The ratio obtained by the empty LL vector was set at 100%, and the relative fold increase of the ratio in the presence of each IRES was set accordingly (Right). The results represent the average of three independent experiments. Asterisks mark statistically significant results. (B) 293 cells were cotransfected with GFP, GFP-DAP5/p86, or GFP-M-FAG/p76 and LL-based bicistronic vectors. Renilla luciferase activity was assessed and normalized to protein and transfection level. The activity obtained by the GFP vector was set at 100%, and the relative activity with GFP-DAP5/p86(GFP86) and GFP-M-FAG/p76(GFP76) was calculated accordingly (Left). The results represent the average of three independent experiments. Protein extract samples of representative experiments were immunoblotted with anti-DAP5 or anti-eIF4GI antibodies (Middle and Right, respectively). The full arrows indicate endogenous DAP5/p97 or eIF4GI. The dashed arrows mark exogenous GFP-DAP5/p86 and GFP-M-FAG/p76. WB, Western blot. (C) 293 cells were cotransfected with GFP, GFP-DAP5/p86, or GFP-M-FAG/p76 and LL-based bicistronic vectors LL-Myc, LL-Apaf-1, and LL-XIAP. The levels of the firefly luciferase were assessed and normalized to protein and transfection levels. For each bicistronic vector, the firefly luciferase activity obtained by the GFP vector was set at 100% (black bar), and relative activities of the GFP-DAP5/p86 (GFP86) and GFP-M-FAG/p76 (GFP76) were calculated accordingly (gray and white bars, respectively). The results of each IRES represent the average of at least three independent experiments. Protein extract samples of representative experiments were immunoblotted with anti-GFP antibodies (Right). (D) 293 cells were transiently transfected with GFP-4GM/p76. Twenty-four hours posttransfection the cells were extracted gently in B buffer. Extract (1.5 mg) was incubated with naked beads as a control, with 7-methyl GTP beads (to trap eIF4E protein, Left) or anti-eIF4A antibodies conjugated to agarose beads (Middle) and washed extensively. Coimmunoprecipitation of GFP-4GM/p76 was assessed by Western blotting the immunoprecipitates with antibodies against the GFP epitope. Alternatively, the ectopically expressed GFP-4GM/p76 was immunoprecipitated (IP) with anti-GFP-conjugated antibodies or naked beads as a control. After resolving the immunoprecipitates on gel, coimmunoprecipitation of endogenous eIF3 was assessed by Western blotting with antibodies against the eIF3/p116 subunit (Right).