3406 Nucleic Acids Research, 2011, Vol. 39, No. 8 of plasmid (28). Then they were centrifuged for 20 min at 4 C and 1570 g to remove cellular debris. The clariﬁed supernatants were concentrated by ultracentrifugation through a 20% sucrose cushion in PBS by using an Optima L-100XP ultracentrifuge and a SW41Ti rotor (Beckman Coulter Inc., Brea, CA, USA) at 151 000 g for 45 min at 4 C. Western blotting analysis Equal amounts of viral lysate from each sample were re- suspended in NuPAGE LDS Sample Buffer (Invitrogen, Carlsbad, CA, USA). Proteins were resolved by SDS– polyacrylamide gels (4–12%) and transferred to nitrocel- lulose membranes by using iBlot (Invitrogen). The mem- branes were treated with mouse anti-MA monoclonal antibody (29), mouse anti-CA monoclonal antibody (Advanced Biotechnologies Inc., Columbia, MD, USA), rabbit anti-NC polyclonal antibody, and rabbit anti-p6 polyclonal antibody (30). ECL Western blotting detection reagents (Nacalai Tesque, Kyoto, Japan) were used for signal detection with horseradish peroxidase conjugated anti-mouse and anti-rabbit IgG (Vector Laboratories, Burlingame, CA, USA). Chemiluminescence was visualized by LAS-1000 (Fujiﬁlm, Tokyo, Japan) accord- ing to the manufacturer’s protocols. Isolation of viral RNA Puriﬁed virions were resuspended in 150 ml of lysis buffer (50 mM Tris–HCl pH 7.5, 100 mM NaCl, 10 mM EDTA, 1% SDS). Viral lysate was treated with 0.2 mg/ml protein- ase K (Invitrogen) at room temperature for 60 min, followed by Tris–EDTA-saturated phenol–chloroform ex- traction, chloroform extraction and ethanol precipitation to collect puriﬁed viral RNA. Native northern blotting analysis Pelleted RNA was resuspended in T-buffer (31), and the thermostability of dimeric viral RNA was determined by incubating RNA aliquots for 10 min at 25, 35, 40, 44, 48 and 52 C (Figure 2), or 25, 35, 40, 45 and 50 C (Figure 3). One quarter of the total amount of puriﬁed viral RNA was applied for each sample. The proportions of the dimers and monomers were measured by electrophoresis at room temperature on a non-denaturing 0.7% agarose gel in 0.5X Tris–borate–EDTA buffer. Field inversion gel electrophoresis was performed for better separation of large RNA molecules. The conditions for ﬁeld inversion gel electrophoresis were as follows: forward, 5 V/cm and 0.6 s; reverse, 5 V/cm and 0.1 s. The agarose gel was then treated with 10% formaldehyde at 65 C for 30 min before Figure 2. Thermal stability of Viral RNA dimers of single Gag-cleavage site mutants. (A) Virion RNA proﬁles detected by northern blotting in a native agarose gel. Viruses were prepared by transfection of 293T cells with the wild-type (WT; pNLNh) virus or its derivative mutants. Aliquots of RNA extracted from virions were resuspended in T-buffer and incubated for 10 min in parallel reactions. The temperatures in which aliquots were incubated are indicated for each lane. (B) A schematic ﬁgure for dimer/monomer calculation. Each lane of the blot was separated into two parts, dimer or larger and monomer or smaller molecules as shown in boxes with dashed lines. The relative amounts of monomeric and total RNAs in each lane were quantitated with ImageGauge, and the percentage of the total represented by monomeric RNA was calculated for each RNA sample. (C) Thermal dissociation kinetics of RNA dimers. Results are the average of three independent experiments. Error bars represent SEM.