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. 2008 Oct 20;29(1):254–265. doi: 10.1128/MCB.01030-08

FIG. 4.

FIG. 4.

PACT and PACTΔ13 induce phosphorylation of PKR in astrocytes. (A) Translation inhibition assay with PACT and PACTΔ13. U251MG cells were transfected with 800 ng of pGL2C (SV40-Luc) 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). Cells were treated with IFN-β and harvested as described in the legend of Fig. 3B. Luc activity was normalized for the total protein present in the extract, and the error bars represent the standard error calculated from two independent experiments performed in triplicate. (B) PACT and PACTΔ13 activate PKR in astrocytes. U251MG cells were transfected alone (lane 1) 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. 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. (C) PACT-induced phosphorylation of PKR in astrocytes is suppressed by TRBP2 overexpression. U251MG cells that overexpress pcDNA3-TRBP2 were transfected alone (lane 1) or with 0.1 (lane 2), 0.5 (lane 3), and 1 (lane 4) μg of pCMV2-Flag-PACT. Whole-cell extracts (150 μg) were subjected to SDS-PAGE and blotted with anti-P-PKR, anti-PKR, anti-Flag, anti-TRBP672, and antiactin antibodies as indicated.