The image contains three panels labeled A, B, and C. Panel A shows a western blot analysis of P K R and e I F 2 phosphorylation with varying levels of A l u single-stranded R N A following poly I:C rechallenge. The blot includes bands for P-P K R, P K R, P-e I F 2 alpha, e I F 2 alpha, and beta-Actin, with different concentrations of A l u s s R N A (0 micrograms, 0.1 micrograms, 1 microgram, 10 micrograms) and with or without poly I:C treatment, as indicated by plus and minus signs. Molecular weight markers (kilodaltons) are shown at 75 and 37. Total P K R and total e I F 2 alpha remain relatively constant, while phosphorylation levels are assessed across conditions. Panel B shows a western blot analysis of wildtype (W T) and D B R 1 knockout (K O) cells transfected with Alu double-stranded R N A. The blot includes bands for P-P K R, P K R, P-e I F 2 alpha, e I F 2 alpha, and beta-Actin, with and without A l u d s R N A transfection, as indicated by plus and minus signs. Molecular weight markers (kilodaltons) are shown at 75 and 37. Phosphorylation of P K R and e I F 2 alpha is compared between W T and D B R 1 K O cells. Panel C shows predicted R N A structures of inverted repeat A l u s from different introns, with numbers indicating the length of the longest continuous duplex regions along the structured segments. The structures are from U S P 8 intron 12, S N A I 2 intron 1, and A C 009784.3 intron 1. Base-paired stem regions and loop structures are depicted schematically along each R N A model.
PKR activation in response to single-stranded and double-stranded Alu RNAs and predicted structures of IR Alus from different introns. (A) High concentrations of single-stranded Alu RNA do not impair PKR activation. Western blot analysis of PKR and eIF2α phosphorylation with varying levels of Alu ssRNA following poly I:C rechallenge. Data are representative of three independent experiments. (B) Reduced PKR and eIF2α phosphorylation in DBR1 KO cells upon Alu dsRNA transfection. WT and DBR1 KO cells were transfected with Alu dsRNA. After 6 h, cell lysates were analyzed by western blot for phosphorylated PKR and eIF2α. Data are representative of three independent experiments. (C) Predicted RNA structures of IR Alus from different introns. Numbers indicate the length of the longest continuous duplex region.