FIG. 9.

Stresses dissociate TRBP-PACT interaction and decrease PACT-induced LTR-Luc expression. (A) Stresses dissociate the TRBP-PACT interaction in HEK293T cells. HEK293T cells were transfected alone (lanes 1, 4, 7, and 11) or with 2 μg of pCMV2-Flag-PACT (lanes 2, 3, 5, 6, 8 to 10, and 12 to 14) and not treated (lanes 1, 2, 4, 5, 7, 8, 11, and 12) or treated with 0.5 mM sodium arsenite (As) for 1 h (lanes 3 and 6) or 10 mM hydrogen peroxide (H₂O₂) for 1 h (lanes 9 and 13) or subjected to serum starvation (SS) for 24 h (lanes 10 and 14). IP was performed with 1.5 mg of protein and anti-Flag antibody. Proteins (150 μg) from each lysate (input; lanes 1 to 3 and 7 to 10) and the immunoprecipitated complexes (lanes 4 to 6 and 11 to 14) were run on a 10% SDS-PAGE gel and blotted using anti-P-PKR, anti-PKR, anti-TRBPjbx, anti-Flag, and antiactin antibodies. Anti-TRBP672 and anti-TRBPjbx were both tested and gave the same results. (B) Stresses decrease PACT-induced LTR-Luc expression. HEK293T cells were transfected alone (lane 1) or with 2 μg of pCMV2-Flag-PACT (lanes 2 to 5) and not treated (lanes 1 and 2) or treated with 0.5 mM sodium arsenite (lane 3) or 10 mM H₂O₂ (lane 4) or subjected to SS (lane 5) as described in panel A. Bars represent the amount of firefly Luc activity per μg of total protein and are the average of three independent experiments (± standard error of the mean). (C) Stresses dissociate the TRBP-PACT interaction in HeLa cells. HeLa cells were transfected alone (lanes 1 and 6) or with 2 μg of pCMV2-Flag-PACT (lanes 2 to 5 and 7 to 10) and not treated (lanes 1, 2, 6, and 7) or treated with 0.5 mM sodium arsenite (lanes 3 and 8) or 10 mM H₂O₂ (lanes 4 and 9) or subjected to SS (lanes 5 and 10) as described in panel A. IP was performed with 1.5 mg of protein and anti-Flag antibody. Proteins (150 μg) from each lysate (input; lanes 1 to 5) and the immunoprecipitated complexes (lanes 6 to 10) were run and blotted as described in panel A.