FIG. 5.

LLnL and doxorubicin both facilitate translocation of rAAV to the nucleus. IB3 cells were infected with AV2eGFP (MOI of 1,000 particles/cell) in the presence or absence of 40 μM LLnL or 1 μM doxorubicin (DOX). At 24 h postinfection, cytoplasmic (Cyt.) and nuclear (Nuc.) fractions were isolated (n = 3 infections for each experimental point). (A) Viral DNA in each fraction was detected by slot blot hybridization against a ³²P-labeled eGFP probe and visualized with a Bio-Rad phosphorimager. (B) Purities of the cytoplasmic and nuclear fractions were confirmed by immunoblotting against the cytoplasmic marker Rab5 and nuclear antigen histone 3. (C) The percentage distributions of the viral genome signals in the nuclear and cytoplasmic fractions were calculated based on the mean (± standard error of the mean) signals for three experimental points. The ³²P signal was quantified with Bio-Rad software.