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. 2009 Aug 5;83(20):10761–10769. doi: 10.1128/JVI.00770-09

FIG. 7.

FIG. 7.

HMW RNA activates MDA5. (A) Amounts of 1 μg and 0.2 μg of the indicated nucleic acid were electrophoretically separated on a 1% agarose gel and subsequently stained with acridine orange. Double-stranded nucleic acid stains green; single-stranded nucleic acid stains red. (B) A 1-μg and 0.2-μg DNA ladder and 1 μg of HeLa-VV RNA were electrophoretically separated at the indicated time points on an agarose gel and stained with acridine orange. (C) A 1-μg DNA ladder and 1 μg of the indicated RNA preparation were electrophoretically separated on an agarose gel and stained with acridine orange. Numbers to the left of the DNA ladders (A to C) show sizes of DNA markers. (D and E) The indicated RNA fraction (see inset of acridine orange gel) was isolated from the agarose gel, and 0.5 or 0.1 μg was used to transfect 3T3 cells. In panel D 3T3 cells were treated with ribavirin to prevent EMCV replication. Graphs show average accumulation + standard deviation of IFN-α/β after overnight culture measured in quadruplicate. (F) Lysate of 293T cells transfected with FLAG-MDA5 for 48 h was used for immunoprecipitation with the K1 (dsRNA) antibody in the absence or presence of 1 μg of the indicated RNA. K1 immunoprecipitates were visualized by Western blotting (WB) with antibody against the FLAG epitope. The arrow points to FLAG-tagged MDA5.