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. 2013 Sep 11;10:96. doi: 10.1186/1742-4690-10-96

Figure 6.

Figure 6

PACT and PACT∆13 are not PKR activators in HIV-producing U251MG astrocytes. A) PACT and PACT∆13 activate PKR and eIF2α in U251MG astrocytes. U251MG astrocytes were mock transfected (lane 1), transfected with 0.5 μg (lane 2 and 5), 1 μg (lane 3 and 6), 2 μg (lane 4 and 7) of pCMV2-Flag-PACT or pCMV2-Flag-PACT∆13 as indicated. 100 μg of cell lysates were resolved by SDS-PAGE and analyzed by immunoblotting using antibodies against P-PKR, PKR, HIV-1 p24, Flag and actin. B) PACT moderately inhibits PKR activation and causes an increase of HIV-1 production in U251MG astrocytes. U251MG astrocytes were mock transfected (lane 1), transfected with 2 μg pNL4-3 (lanes 2–5) and 0.5, 1 and 2 μg of pCMV2-Flag-PACT (lanes 3–5). The corresponding empty plasmid, pCMV2, was supplemented to each transfection to have the same amount of DNA transfected in each well. (Top) The RT activity is displayed as a percentage of activation compared to pNL4-3 alone. Results shown are an average of 3 independent transfections ± SEM. (Bottom) Cell extracts were immunoblotted and probed using antibodies against P-PKR, PKR, Flag, HIV-1 p24, P-eIF2α, eIF2α and actin. Shown is a representative experiment. C) PACT∆13 affects neither PKR activation nor HIV-1 production in U251MG astrocytes. U251MG astrocytes were mock transfected (lane 1), transfected with 2 μg pNL4-3 (lanes 2–5) and 0.5, 1 and 2 μg of pCMV2-Flag-PACT13 (lanes 3–5). The corresponding empty plasmid, pCMV2, was supplemented to each transfection to have the same amount of DNA transfected in each well. (Top) The RT activity was displayed as a percentage of activation compared to pNL4-3 alone. Results shown are an average of 3 independent transfections ± SEM. (Bottom) Cell extracts were immunoblotted and probed using antibodies against P-PKR, PKR, Flag, HIV-1 p24, P-eIF2α, eIF2α and actin. Shown is a representative experiment.