Nucleic Acids Research, 2011, Vol. 39, No. 8 3407 Figure 3. The mutants to mimic sequential processing of HIV-1 Gag. (A) Schematic representations of the mutants. CA/p2 and NC/p1 mutants were added for reference. Shaded boxes represent fusion proteins which resulted from the mutations. (B) Detection of HIV-1 protein produced in pelleted virions by western blotting with various anti-Gag antibodies. Positions of Gag proteins and precursors are indicated. (C) Raw virion RNA proﬁles detected by northern blotting in a native agarose gel as in Figure 2. (D) Thermal dissociation kinetics of RNA dimers. The experiments were similarly performed as described in Figure 2. Results are the average of three to ﬁve independent experiments. Error bars represent SEM. being washed with H2O three times, and the RNA was blotted electrically onto a Hybond-N+ nylon membrane (GE healthcare, Chalfont St Giles, UK). RNA northern hybridization analysis was performed as described previously (31). The plasmid pT7pol (31) and T7 RNA polymerase (Takara Bio Inc., Shiga, Japan) were used to synthesize the riboprobe, which speciﬁcally detects unspliced viral RNAs, for northern hybridization. Hybridization was carried out in the presence of Rapid- Hyb buffer (GE healthcare). Membranes were washed extensively with 0.1 SSC (1 SSC was comprised of 15.0 mM NaCl and 15 mM sodium citrate [pH 7.0]) 0.1% SDS at 70 C. In experiments designed to assess the conversion of dimers to monomers, the relative amounts of both RNA species were quantitated with a FLA5000 image analyzer and BAS imaging plate (Fujiﬁlm). To characterize the proﬁle of genomic RNA, a lane on the blot was scanned by ImageGauge software (Fujiﬁlm) and the signal intensity and distance migrated from each well were measured.