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Figure 5 | BMC Microbiology

Figure 5

From: Phenotypic silencing of cytoplasmic genes using sequence-specific double-stranded short interfering RNA and its application in the reverse genetics of wild type negative-strand RNA viruses

Figure 5

Induction of target mRNA degradation but not interferon response by dsRNA. Panel A: Semi-quantitative RT-PCR to measure the indicated RSV gene mRNA and genomic RNA in A549 cells were performed as described under Materials and Methods. Where indicated (labeled '+'), anti-F dsRNA was used at a concentration of 20 nM. PCR samples were taken at the end of the number of cycles indicated on top (20, 22, 24, and 26). Actin mRNA was also quantitated as a control. Note that in dsRNA-untreated cells (labeled '-') the F band is visible even at 20 cycles, whereas in the treated cells, appearance of a comparable intensity required 4 additional PCR cycles, i.e., 16-fold more amplification. Panel B: Assay of eIF-2α phosphorylation. Metabolic 32P-labeling and immunoprecipitation (IP) analysis of eIF-2 have been described in Materials and Methods. An autoradiograph of the gel is shown. The cells were treated with no RNA (lane C), 100 nM thapsigargin (lane 1), 100 nM A23178 (lane 2), 50 nM anti-P dsRNA (lane 3), or 50 nM anti-F dsRNA (lane 4). Note the increased phosphorylation of eIF-2 in lanes 1 and 2 only. The immunoblot (IB) shows that the total amount of eIF-2α protein was not affected by the treatments.

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