Subcellular localization of Gag and the gRNA. Subcellular fractionations of 293T cells expressing wild-type HIV-1 (A, as in (21)) or NC(ΔZF1ZF2) (B) or ΔNC (C) were analyzed by OptiPrep gradient centrifugation. Cells were broken as described in materials and methods and the post-nuclear supernatant (PNS) was fractionated by Optiprep gradient. 20 μl of each fraction were loaded on SDS-PAGE, and Gag and Lamp2 were analyzed by immunoblotting using anti-Cap24 and anti-Lamp2 antibodies. Each fraction of the gradient was tested for the presence of the gRNA by RT-PCR as described in materials and methods. The expected 132 bp DNA fragment was detected on 1% agarose gel. In addition to the gradient analyses, the immunofluorescence (IF) detections are shown, representing the cells stained with an anti-CAp24 (in green) for Gag (A) or mutated Gag (B and C), and the FISH treatment of the 293T cells expressing HIV-1 (A) or the NC Gag mutants (B and C) for the gRNA using the Gag-oligo-Cy3 probe (in red).