FIG. 3.

PACT activation of PKR in HeLa cells requires deletion of 13 amino acids (Δ13) and IFN induction. (A) Schematic representation of PACT and PACTΔ13 and their domains. Domain C is also called the Medipal domain of PACT by homology with TRBP (46). (B) Translation inhibition assay with PACT and PACTΔ13. (Top) HeLa cells were transfected with 800 ng of pGL2C (Promega) encoding Luc (SV40-Luc) and without plasmid (lane 1) or with 0.15 μg (lane 2), 0.3 μg (lane 3), 0.45 μg (lane 4), or 0.6 μg (lane 5) of Flag-PACT-pCB6+ (light bars) or Flag-PACTΔ13-pCB6+ (dark bars). At 24 h posttransfection, cells were treated with 100 U/ml of IFN-β for 24 h and then harvested for Luc activity measurement. Luc activity was normalized for the total protein present in the extract, and the error bars represent the standard error calculated from six independent values. (Bottom) Extracts from lane 1 (no PACT) above or lane 5 (PACT or PACTΔ13) were separated by SDS-PAGE and blotted with an anti-Flag or antiactin antibody. (C) PACT or PACTΔ13 does not activate PKR in the absence of IFN. HeLa cells were transfected alone (lanes 1, 8, and 9) or with 0.1 (lanes 2 and 5), 0.5 (lanes 3 and 6), and 1 (lanes 4 and 7) μg of pCMV2-Flag-PACT or pCMV2-Flag-PACTΔ13, as indicated, or with 2 μg of pcDNA1-PKR and 0.02 μg of poly(I)·poly(C) (lane 9). Whole-cell extracts (200 μg) were subjected to SDS-PAGE and blotted with anti-P-PKR, anti-PKR, anti-Flag, and antiactin antibodies as indicated.