FIG. 8.

Effects of flAlu RNA overexpression on PKR activity. (A) Aliquots of immunoprecipitates from cells transfected with clone XAT as described in the legend to Fig. 3B were tested for PKR autophosphorylation. Immunoprecipitated samples were from 293 cells transfected with pUC (293; lane 1) and with 2.5, 5, and 10 μg of clone XAT (Alu; lanes 2 through 4, respectively). By phosphorimager analysis, the levels of PKR activity were 82% (lane 2) and 26% (lanes 3 and 4) of the activity in the control (lane 1). (B) The levels of PKR in the same immunoprecipitates were tested by Western analysis with a previously described antibody (4). The samples were from 293 cells transfected with pUC as a control (293; lane 3) and with 2.5, 5, and 10 μg of clone XAT (Alu; lanes 4 through 6, respectively). As additional controls, cells overexpressing recombinant PKR (Rec; lane 2) and a mock immunoprecipitation (Mock; lane 1) were examined. By using a lower percentage of acrylamide and running the gel longer, a doublet band of PKR was revealed as discussed in the text.