Radprime

Genomic DNA of wild-type and CHAPb Tg mouse tails was extracted in 0.5 ml tail lysisbuffer (50 mM Tris pH 8.0, 100 mM EDTA pH 8.0, 100mM NaCl, 1% SDS and 10 mg/ml prot K) at 55 ^oC overnight. DNA was precipitated by phenol-chloroform extraction and 10 μg DNA was digested by BamHI (Promega) or XmnI (New England Biolabs) overnight and run on a 1% agarose gel. Probes were generated using the following primers: 5’- AGGGGTCCAGCTCTTTGAAC-3’ and 5’-AGGCTTAAAGCGTCCTCCTC-3’. The PCR products were then radioactively labelled using α-[p32]dATP (PerkinElmer) by random priming (RadPrime, Invitrogen). DNA blots (Hybond-N+, GE Healthcare) were hybridized with the radioactive probe in ExpressHyb Hybridization buffer (Clontech) and visualized by using a Phosphorimager.

Total RNA was extracted from cells using TRIzol. 15μg RNA in RNA loading buffer (32% formamide, 1x MOPS-EDTA-Sodium acetate (MESA, Sigma) and 4.4% formaldehyde) was denatured at 65°C for 10 min and resolved on a 1% agarose gel containing 1x MESA and 3.7% formaldehyde. The RNA was transferred and UV crosslinked to a Zeta-probe membrane (Bio-Rad). Transfer efficiency was assessed by visualizing ribosomal RNA on the membrane using methylene blue stain. The membrane was destained and hybridized with α-³²P dATP labelled DNA probes (RadPrime, Invitrogen) complementary to the DENV 3’UTR at 65°C for 3 hrs using ExpressHyb hybridization buffer (Clontech). Autoradiographs were quantified using ImageQuant (GE Healthcare).